lysates Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 80
    Millipore berghei lysate
    Infected red blood cells (iRBCs) loads in the lung are associated with the development of ALI/ARDS in PbA-infected DBA/2 mice. (A) Survival curve and (B) parasitemia of DBA/2 mice infected with 10 6 P . <t>berghei</t> ANKA iRBCs. The gray frame shows the period between the 7 th and 12 th days post-infection (dpi) where approximately 60% of the animals died with signs of ALI/ARDS. (C) Enhanced respiratory pause (Penh) and (D) respiratory frequency (RF), as well as (E) parasitemia were measured on the 7 th dpi. Data are representative of five independent experiments and are expressed as the mean ± SEM (Mann-Whitney test or Kruskal-Wallis test; n = 10–12 mice/experiment; *p
    Berghei Lysate, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/berghei lysate/product/Millipore
    Average 80 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    berghei lysate - by Bioz Stars, 2019-05
    80/100 stars
      Buy from Supplier

    79
    Thermo Fisher translationally active hela cell lysate
    DON induces transient <t>PKR</t> activation in <t>HeLa-based</t> cell-free system. ( A ) DON (100, 250, 1000 ng/mL), poly (IC) (100 ng/mL) and/or PKR inhibitors, 2-AP (2 mM) and C16 (2 µM), were added to a cell-free system comprised of HeLa-based cell-derived, translationally active cell-free system containing ribosomes and ATP but devoid of cell membrane, nuclei, mitochondria, DNA and mRNA. After incubation for 20 min at 30 °C, Western analysis was conducted with PKR and p-PKR antibodies; ( B ) HeLa-based cell-free assay mixtures were incubated with DON (250 ng/mL) for indicated time intervals and subjected to Western blotting with PKR, p-PKR and eIF2α, p -eIF2α antibodies. Data are representative of at least three replicate experiments.
    Translationally Active Hela Cell Lysate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/translationally active hela cell lysate/product/Thermo Fisher
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    translationally active hela cell lysate - by Bioz Stars, 2019-05
    79/100 stars
      Buy from Supplier

    91
    ZeptoMetrix ebv lysate
    Clonotypic distribution of T cells comparing <t>EBV</t> − HIV + versus EBV + HIV + patients under EBV-stimulated conditions. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro with EBV lysate. The distribution of <t>CD4</t> + (A) and CD8 + (B) T cells positive for TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.
    Ebv Lysate, supplied by ZeptoMetrix, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ebv lysate/product/ZeptoMetrix
    Average 91 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    ebv lysate - by Bioz Stars, 2019-05
    91/100 stars
      Buy from Supplier

    88
    Bio-Rad foxj1 doublet lysates
    IKK-inhibiting viral degradation of <t>Foxj1</t> and CSF/brain barrier disruption. a IHC staining of primary EC cultures without (Ctrl) or with indicated treatment conditions, labeled with Foxj1 and VP16 antibodies, and DAPI. Cultures were treated with viruses for 16 h. Quantifications showing Foxj1 + cells per total DAPI nuclei in treatment conditions as percentage of Ctrl condition in each experiment (read dashed line). * P
    Foxj1 Doublet Lysates, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/foxj1 doublet lysates/product/Bio-Rad
    Average 88 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    foxj1 doublet lysates - by Bioz Stars, 2019-05
    88/100 stars
      Buy from Supplier

    86
    Becton Dickinson rotavirus infected cell lysate
    Schematic representation of Ar/NH 3 plasma-treated MNBs and their binding to anti-virus antibody. Notes: Graphite-encapsulated MNBs were treated with ammonia plasma and modified with amino groups. SPDP was reacted with the amino group of modified MNBs (NH 2 -beads) at pH 7–8. <t>Anti-rotavirus</t> antibody or anti-dengue virus antibody was reduced using DTT, resulting in the breakage of S-S bonds and the generation of S-H groups. The S-H group of the antibody was then further reacted with SPDP-NH-MNBs. The resultant MNBs are termed antibody-integrated MNBs. Abbreviations: DTT, dithiothreitol; MNBs, magnetic nanobeads; SPDP, N -succinimidyl 3-(2-pyridyldithio)propionate.
    Rotavirus Infected Cell Lysate, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rotavirus infected cell lysate/product/Becton Dickinson
    Average 86 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    rotavirus infected cell lysate - by Bioz Stars, 2019-05
    86/100 stars
      Buy from Supplier

    80
    Greenlee purkinje cell lysates
    Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within <t>Purkinje</t> cells and IgG-mediated cytotoxicity. In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2 ). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.
    Purkinje Cell Lysates, supplied by Greenlee, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purkinje cell lysates/product/Greenlee
    Average 80 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    purkinje cell lysates - by Bioz Stars, 2019-05
    80/100 stars
      Buy from Supplier

    80
    Promega hela lysates
    Effects of <t>DOC-1R</t> expression on the regulation of G1 phase-related gene expressions. (A) Overexpression of DOC-1R. pFLAG-DOC-1R was transfected into <t>HeLa</t> cells and 48 h after transfection, cells were extracted and subjected to Western blot analysis of CDK2, cyclin D1, cyclin E, p21, p27 and p53 protein, respectively. (B) Knockdown of endogenous DOC-1R. DOC-1R shRNA plasmid was transfected into HEK-293 cells and 48 h post-transfection, cells were extracted and subjected to Western blot analysis of related proteins. A scrambled shRNA plasmid was used as a control.
    Hela Lysates, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela lysates/product/Promega
    Average 80 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    hela lysates - by Bioz Stars, 2019-05
    80/100 stars
      Buy from Supplier

    79
    Luminex pneumoniae lysates
    IVIG causes aggregation of both encapsulated and unencapsulated S . <t>pneumoniae</t> . ( A ) Light microscopy of bacteria (at 100 X with rapid Romanovsky stain) after 8 hr culture of wild-type (TIGR4) and unencapsulated (TIGR4Δcps) bacteria with or without 10% IVIG. (B) Effect of relative concentration of IVIG on size of bacterial aggregate particles after 30 minute incubation of wild-type (open bars) or unencapsulated (filled bars) TIGR4 measured as forward scatter in flow cytometry. P values were calculated using one-way ANOVA. (C) to (F) Changes in optical density at 580nm (OD 580 ) during culture of wild-type (triangles) and unencapsulated (circles) S . pneumoniae (C) TIGR4, (D) ST2 (D39), (E) ST3 (0100993), (F) or ST23F strain in THY broth supplemented with either 10% IVIG (filled symbols) or PBS (empty symbols). (G) Effect of vigorous pipetting (filled symbols) or no pipetting (empty symbols) immediately prior to measurement of OD580 during culture of wild-type (triangles) and unencapsulated (circles) TIGR4 in 10% IVIG. (H) Effect of addition of either 1% IVIG (filled circles) or 10% (upside triangles), 5% (diamonds) or 2% (crosses) papain-treated IVIG, or PBS (empty circles) on OD 580 during culture of unencapsulated TIGR4. For panels B to H, data are presented as means and error bars represent SDs of three to four technical replicates and are representative of experiments repeated at least twice. *, P
    Pneumoniae Lysates, supplied by Luminex, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pneumoniae lysates/product/Luminex
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pneumoniae lysates - by Bioz Stars, 2019-05
    79/100 stars
      Buy from Supplier

    79
    Enzo Biochem sh sy5y cells lysates
    Effects of CSZ on phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK and cyclic adenosine monophosphate response element-binding protein (CREB) in Aβ-stimulated <t>SH-SY5Y</t> cells. Phosphorylation of ERK1/2, p38 MAPK and CREB were examined using ELISA kit described in “Materials and Methods” section. (A) ERK1/2 phosphorylation. (B) p38 MAPK phosphorylation. SH-SY5Y cells were incubated with or without pretreatment with CSZ for 1 h followed by treatment with Aβ for 30 min. The effects of respective inhibitor were also examined. (C) CREB phosphorylation. Values are expressed as mean + SEM # Compared vs. respective control cells ( p
    Sh Sy5y Cells Lysates, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sh sy5y cells lysates/product/Enzo Biochem
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    sh sy5y cells lysates - by Bioz Stars, 2019-05
    79/100 stars
      Buy from Supplier

    79
    Promega hek293 cell lysates
    The effect of 3′ overhang on RISC function in Dicer kd, Ago2 kd and TRBP kd cells. Efficiencies of target downregulation by four variants of EGFPS1A siRNA ( Figure 1 ) were evaluated using Dual Luciferase assays with siDicer, siAgo2 or siTRBP treated <t>HEK293</t> cells. To determine effects of Dicer, Ago2 or TRBP knockdowns on the silencing activity of the siEGFPS1A variants described in Figure 1 , HEK293 cells were treated with 40 nM siDicer, siAgo2 or siTRBP for 2 days prior to the co-transfection. For the second round of transfection, Dicer kd, Ago2 kd or TRBP kd cells were transfected with the psi-EGFP-S1 sense reporter and 200 pM siEGFPS1A variants. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from four co-transfections).
    Hek293 Cell Lysates, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek293 cell lysates/product/Promega
    Average 79 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    hek293 cell lysates - by Bioz Stars, 2019-05
    79/100 stars
      Buy from Supplier

    79
    Thermo Fisher pgrmc2 lysates
    <t>PGRMC2</t> interacts with ALADIN determined by IP-western and reciprocal IP- w estern assays. (A) Whole cell lysates of GFP-ALADIN, PGRMC2-GFP- and GFP- (as negative control) expressing NCI-H295R cells were used and GFP pulldown performed followed by western blot with indicated antibodies. PGRMC2 (24 kDa, arrow) could be detected after GFP-ALADIN (86 kDa) pulldown. ALADIN (59 kDa, arrow) and PGRMC2 could be both detected after PGRMC2-GFP (51 kDa) pulldown. GFP (27 kDa) was ascertained after GFP control pulldown but the control remained empty for PGRMC2 and ALADIN. (B) Whole cell lysates of NCI-H295R cells were used for ALADIN and PGRMC2 pulldown. Normal mouse (m IgG) and rabbit IgGs (Rb IgG) served as negative control. Western blot was performed with indicated antibodies. Non-bound (NB) IP fractions are also shown for each pulldown and bound IP eluates are separated by one lane each from the specific controls to eliminate false positive detection. PGRMC2 (arrow) precipitated in endogenous ALADIN pulldown. Control m IgG pulldown remained empty. ALADIN (arrow) reciprocally precipitated after PGRMC2 pulldown. Control Rb IgG pulldown remained empty showing a cross-reactive band a few kDa higher than ALADIN.
    Pgrmc2 Lysates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgrmc2 lysates/product/Thermo Fisher
    Average 79 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    pgrmc2 lysates - by Bioz Stars, 2019-05
    79/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc hela cell lysates
    Extent of G 2 arrest is enhanced in <t>E4-17/16K-expressing</t> cells by overexpression of Wee1. (A) Western blot analysis of Wee1 protein levels in <t>HeLa</t> cells either mock transfected (−) or transfected (+) with Wee1 expression plasmid. Fluorescence
    Hela Cell Lysates, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela cell lysates/product/Cell Signaling Technology Inc
    Average 78 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    hela cell lysates - by Bioz Stars, 2019-05
    78/100 stars
      Buy from Supplier

    78
    NuSep femx i total cell lysates
    Differences in lipid profiling between prom1-exo (MV) and parental <t>FEMX-I</t> cells (FEMX). An automated ESI-tandem mass spectrometry approach was used for lipid profiling. Averages of three to five determinations for each sample group were calculated. Red, lipid species over-expressed in prom1-exo; blue, lipid species over-expressed in parental FEMX-I cells. *, p
    Femx I Total Cell Lysates, supplied by NuSep, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/femx i total cell lysates/product/NuSep
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    femx i total cell lysates - by Bioz Stars, 2019-05
    78/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc cell lysate binding assays rabbit anti grb2
    Phosphopeptide microarray analysis of human cardiomyocyte progenitor cells showing detection of multiple proteins <t>(GRB2</t> and SRC) on the same phosphoprotein-protein interaction complexes. The red spots refer to the presence of GRB2 protein (Rabbit Anti GRB2 with Rabbit anti-IgG conjugated with Alexa 647); The green spots refer to SRC (Mouse Anti SRC with Mouse anti-IgG conjugated with Alexa 594); The yellow spots is the results of merge of GRB2 and SRC images together showing the presence of GRB2 and SRC on the same phosphopeptide-protein complex.
    Cell Lysate Binding Assays Rabbit Anti Grb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell lysate binding assays rabbit anti grb2/product/Cell Signaling Technology Inc
    Average 78 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    cell lysate binding assays rabbit anti grb2 - by Bioz Stars, 2019-05
    78/100 stars
      Buy from Supplier

    77
    Novus Biologicals adult human skeletal muscle whole lysates
    Panx1 and Panx3 are expressed in the <t>skeletal</t> <t>muscle</t> tissue. A , expression of Panx1, Panx2, and Panx3 proteins in <t>adult</t> <t>human,</t> mouse, and rat skeletal muscle <t>whole</t> tissue <t>lysates</t> was analyzed by Western blotting. Various molecular weight species of Panx1 and Panx3 were expressed, whereas Panx2 was absent or below detectable levels. HEK293T cells transfected with Panx1, Panx2, or Panx3 were used as positive controls. Desmin is a muscle-specific protein. B , human skeletal muscle tissue in skin samples was labeled for Panx1 (labeled in red ) and Panx3 (labeled in red ). Representative images are shown. Panx1 was detected as a punctate stain, whereas Panx3 was observed as diffuse labeling. Higher magnification micrographs of Panx3 labeling show a striated pattern. Peptide competition revealed loss of specific staining. Blue = nuclei; bars = 50 μm.
    Adult Human Skeletal Muscle Whole Lysates, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adult human skeletal muscle whole lysates/product/Novus Biologicals
    Average 77 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    adult human skeletal muscle whole lysates - by Bioz Stars, 2019-05
    77/100 stars
      Buy from Supplier

    77
    Novus Biologicals hif 2α standard lysate
    Inhibition of both HIF1α and <t>HIF-2α</t> diminishes Bcl-2 levels without affecting BAX levels. Cell lysates collected from cells treated with 0.5 µM, 5 µM, and 50 µM of HIF-1α/HIF-2α translation inhibitors were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX, and a significant decrease in the levels of Bcl-2 coupled with the loss of VEGF ( Figure 4L ) was observed with the double translation inhibitor ( A ). B and C represent the corresponding densitometry analysis.
    Hif 2α Standard Lysate, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hif 2α standard lysate/product/Novus Biologicals
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    hif 2α standard lysate - by Bioz Stars, 2019-05
    77/100 stars
      Buy from Supplier

    76
    Proteome Sciences Inc raw264 7 cell lysates
    Outputs of proteomic analysis for CA-altered proteins. (A) Venn diagram showing numerical distribution of proteins identified in different <t>RAW264.7</t> cell lysates by the nanoLC-LTQ-Orbitrap MS/MS approach. Cells were treated with vehicle (control group), LPS (1 μg/ml, LPS group), and LPS with 20 μM of CA (LPS+CA group) for 6 h, respectively. (B–D) Proteins significantly up-regulated and down-regulated by CA were classified into different cellular components (B) , molecular functions (C) , and BPs (D) .
    Raw264 7 Cell Lysates, supplied by Proteome Sciences Inc, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raw264 7 cell lysates/product/Proteome Sciences Inc
    Average 76 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    raw264 7 cell lysates - by Bioz Stars, 2019-05
    76/100 stars
      Buy from Supplier

    Image Search Results


    Infected red blood cells (iRBCs) loads in the lung are associated with the development of ALI/ARDS in PbA-infected DBA/2 mice. (A) Survival curve and (B) parasitemia of DBA/2 mice infected with 10 6 P . berghei ANKA iRBCs. The gray frame shows the period between the 7 th and 12 th days post-infection (dpi) where approximately 60% of the animals died with signs of ALI/ARDS. (C) Enhanced respiratory pause (Penh) and (D) respiratory frequency (RF), as well as (E) parasitemia were measured on the 7 th dpi. Data are representative of five independent experiments and are expressed as the mean ± SEM (Mann-Whitney test or Kruskal-Wallis test; n = 10–12 mice/experiment; *p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Infected red blood cells (iRBCs) loads in the lung are associated with the development of ALI/ARDS in PbA-infected DBA/2 mice. (A) Survival curve and (B) parasitemia of DBA/2 mice infected with 10 6 P . berghei ANKA iRBCs. The gray frame shows the period between the 7 th and 12 th days post-infection (dpi) where approximately 60% of the animals died with signs of ALI/ARDS. (C) Enhanced respiratory pause (Penh) and (D) respiratory frequency (RF), as well as (E) parasitemia were measured on the 7 th dpi. Data are representative of five independent experiments and are expressed as the mean ± SEM (Mann-Whitney test or Kruskal-Wallis test; n = 10–12 mice/experiment; *p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Infection, Mouse Assay, MANN-WHITNEY

    CXCR4 antagonist treatment protects DBA/2 mice from ALI/ARDS. (A) Survival and (B) parasitemia curves in P . berghei ANKA-infected mice treated with 5 mg/kg of AMD3100 or DMSO + saline solution (control group) on the 1 st , 3 rd , 5 th and 7 th days post-infection (dpi). (C) Lung tissues from untreated mice (CTR) (died on the 9 th dpi with ALI/ARDS) and AMD3100-treated mice (died on the 21 st dpi due to hyperparasitemia) (400x, scale bar 25 μm). (D) Number of neutrophils in the lungs, measured via flow cytometry, from infected AMD3100-treated mice and control mice (CTR). (E) Enhanced respiratory pause (Penh) and (F) respiratory frequency (RF) data for these mice. Data are representative of three independent experiments and are expressed as the mean ± SEM. The data from (D) to (F) were collected on the 7 th dpi. (A, log-rank test and Wilcoxon-Gehan-Breslow test, p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: CXCR4 antagonist treatment protects DBA/2 mice from ALI/ARDS. (A) Survival and (B) parasitemia curves in P . berghei ANKA-infected mice treated with 5 mg/kg of AMD3100 or DMSO + saline solution (control group) on the 1 st , 3 rd , 5 th and 7 th days post-infection (dpi). (C) Lung tissues from untreated mice (CTR) (died on the 9 th dpi with ALI/ARDS) and AMD3100-treated mice (died on the 21 st dpi due to hyperparasitemia) (400x, scale bar 25 μm). (D) Number of neutrophils in the lungs, measured via flow cytometry, from infected AMD3100-treated mice and control mice (CTR). (E) Enhanced respiratory pause (Penh) and (F) respiratory frequency (RF) data for these mice. Data are representative of three independent experiments and are expressed as the mean ± SEM. The data from (D) to (F) were collected on the 7 th dpi. (A, log-rank test and Wilcoxon-Gehan-Breslow test, p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Mouse Assay, Infection, Flow Cytometry, Cytometry

    Early depletion of neutrophils protects DBA/2 mice from ALI/ARDS. (A) Survival and (B) parasitemia curves in P . berghei ANKA-infected mice treated with 0.2 mg/kg of anti-GR1 or IgG antibodies (control group) on the 1 st day post-infection (dpi). Respiratory parameters (C) enhanced pause (Penh) and (D) respiratory frequency on the 7 th dpi. (E) Photomicrographs of lung tissue on the day of death from non-infected mice, control mice (8 th dpi) and anti-GR1 treated mice (18 th dpi) (400x, scale bar 25 μm). Number/frequency of neutrophils in the blood from infected control (black) and anti-GR1-treated (gray) mice measured via (F) flow cytometry and (G) an analysis of the blood smears. Data are representative of three independent experiments and are expressed as the mean ± SEM (A, log-rank test and Wilcoxon-Gehan-Breslow test, p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Early depletion of neutrophils protects DBA/2 mice from ALI/ARDS. (A) Survival and (B) parasitemia curves in P . berghei ANKA-infected mice treated with 0.2 mg/kg of anti-GR1 or IgG antibodies (control group) on the 1 st day post-infection (dpi). Respiratory parameters (C) enhanced pause (Penh) and (D) respiratory frequency on the 7 th dpi. (E) Photomicrographs of lung tissue on the day of death from non-infected mice, control mice (8 th dpi) and anti-GR1 treated mice (18 th dpi) (400x, scale bar 25 μm). Number/frequency of neutrophils in the blood from infected control (black) and anti-GR1-treated (gray) mice measured via (F) flow cytometry and (G) an analysis of the blood smears. Data are representative of three independent experiments and are expressed as the mean ± SEM (A, log-rank test and Wilcoxon-Gehan-Breslow test, p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Mouse Assay, Infection, Flow Cytometry, Cytometry

    Plasmodium berghei -iRBCs promote more NETs than RBCs in mouse neutrophils. (A) Immunofluorescence of non-stimulated (NS) DBA/2 mouse neutrophils and of neutrophils stimulated with PMA [positive control (PC)], red blood cells (RBCs), and P . berghei ANKA (iRBCs) stained with Sytox Green (630x, scale bar 20 μm). (B) Quantification of extracellular DNA (NETs) using Sytox Green based on units of arbitrary fluorescence (UAF). Measurements were performed after 60, 120 and 180 minutes of stimulation. The data are representative of three independent experiments with mean ± SEM (Kruskal-Wallis test where *** p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Plasmodium berghei -iRBCs promote more NETs than RBCs in mouse neutrophils. (A) Immunofluorescence of non-stimulated (NS) DBA/2 mouse neutrophils and of neutrophils stimulated with PMA [positive control (PC)], red blood cells (RBCs), and P . berghei ANKA (iRBCs) stained with Sytox Green (630x, scale bar 20 μm). (B) Quantification of extracellular DNA (NETs) using Sytox Green based on units of arbitrary fluorescence (UAF). Measurements were performed after 60, 120 and 180 minutes of stimulation. The data are representative of three independent experiments with mean ± SEM (Kruskal-Wallis test where *** p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Immunofluorescence, Positive Control, Staining, Fluorescence

    Neutrophil interactions in malaria-associated ALI/ARDS. Following P . berghei ANKA infection in DBA/2 mice, neutrophils promote the pathogenesis of ALI/ARDS. In particular, the release of myeloperoxidase and reactive species of oxygen and the formation of neutrophil extracellular traps determines the cause of death of these mice.

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Neutrophil interactions in malaria-associated ALI/ARDS. Following P . berghei ANKA infection in DBA/2 mice, neutrophils promote the pathogenesis of ALI/ARDS. In particular, the release of myeloperoxidase and reactive species of oxygen and the formation of neutrophil extracellular traps determines the cause of death of these mice.

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Infection, Mouse Assay

    Recombinant human DNase 1 (Pulmozyme) and an elastase inhibitor (Sivelestat) improve outcomes of malaria-associated ALI/ARDS. (A) Lung sections of DBA/2 mice infected with 10 6 P . berghei ANKA-iRBCs on the day of death (9 th dpi) were stained for histone (red), myeloperoxidase (MPO) (green), and DNA (blue). Overlapping colors indicate cloud-like structures. White arrowheads show selected cells forming NETs (400x, scale bar 25 μm). (B) Survival, (C) parasitemia, (D) enhanced respiratory pause (Penh) and (E) respiratory frequency (RF) on the 7 th days post-infection (dpi) in P . berghei ANKA-infected mice treated with Pulmozyme (50 μg/mouse) or saline solution (control group) on the 3 rd and 6 th dpi. Data are representative of three independent experiments. (F) Survival, (G) parasitemia, (H) enhanced respiratory pause (Penh) and (I) respiratory frequency (RF) on the 7 th dpi in P . berghei ANKA-infected mice treated with elastase inhibitor (30 mg/kg) or saline solution (control group) on the 3 rd and 6 th dpi. Data are representative of one experiment. (J) Lung tissue on the day of death from representative control mice (died on the 10 th dpi with ALI/ARDS), Pulmozyme-treated mice (died on the 20 th dpi), and Sivelestat-treated mice (died on the 20 th dpi) stained with HE (400x, scale bar 25 μm). Data are expressed as the mean ± SEM (C, log-rank test and Wilcoxon-Gehan-Breslow, p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Recombinant human DNase 1 (Pulmozyme) and an elastase inhibitor (Sivelestat) improve outcomes of malaria-associated ALI/ARDS. (A) Lung sections of DBA/2 mice infected with 10 6 P . berghei ANKA-iRBCs on the day of death (9 th dpi) were stained for histone (red), myeloperoxidase (MPO) (green), and DNA (blue). Overlapping colors indicate cloud-like structures. White arrowheads show selected cells forming NETs (400x, scale bar 25 μm). (B) Survival, (C) parasitemia, (D) enhanced respiratory pause (Penh) and (E) respiratory frequency (RF) on the 7 th days post-infection (dpi) in P . berghei ANKA-infected mice treated with Pulmozyme (50 μg/mouse) or saline solution (control group) on the 3 rd and 6 th dpi. Data are representative of three independent experiments. (F) Survival, (G) parasitemia, (H) enhanced respiratory pause (Penh) and (I) respiratory frequency (RF) on the 7 th dpi in P . berghei ANKA-infected mice treated with elastase inhibitor (30 mg/kg) or saline solution (control group) on the 3 rd and 6 th dpi. Data are representative of one experiment. (J) Lung tissue on the day of death from representative control mice (died on the 10 th dpi with ALI/ARDS), Pulmozyme-treated mice (died on the 20 th dpi), and Sivelestat-treated mice (died on the 20 th dpi) stained with HE (400x, scale bar 25 μm). Data are expressed as the mean ± SEM (C, log-rank test and Wilcoxon-Gehan-Breslow, p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Recombinant, Mouse Assay, Infection, Staining

    Neutrophils are involved in the pathogenesis of malaria-associated ALI/ARDS. DBA mice were infected with 10 6 P . berghei ANKA-iRBCs. (A) Representative lung photomicrograph (630x, scale bar 10 μm) of tissue from a mouse with ALI/ARDS stained with HE, highlighting the inflammatory infiltrate, especially of neutrophils (arrows and insert) on the day of death (11 th day post-infection (dpi). (B) Neutrophil number (Ly6G + CD11b + ) and (C) Ncf2 gene expression in the lungs. (D) KC (CXCL-1) and (E) MIP-2 (CXCL-2) in the sera of mice with ALI/ARDS (red) and HP (black) on the 3 rd and 5 th dpi. Number of neutrophils (Ly6G + CD11b + ) producing (F) reactive oxygen species (DHR + ) in the lungs or producing myeloperoxidase (MPO) in the (G) lungs and (H) bronchoalveolar lavage fluid (BALF) of mice with ALI/ARDS compared with those from mice with HP. (I e J) Leukocytes in the BALF of mice. Arrowheads indicate neutrophils (400x, scale bar 25 μm). Data for three grouped experiments are expressed as the mean ± SEM. The data from figures B, C, F, G, H, I and J were collected on the 7 th dpi. (Mann-Whitney test and Kruskal-Wallis test; (n = 5); ALI/ARDS: n = 12–20, HP: n = 10–15; * p

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Neutrophils are involved in the pathogenesis of malaria-associated ALI/ARDS. DBA mice were infected with 10 6 P . berghei ANKA-iRBCs. (A) Representative lung photomicrograph (630x, scale bar 10 μm) of tissue from a mouse with ALI/ARDS stained with HE, highlighting the inflammatory infiltrate, especially of neutrophils (arrows and insert) on the day of death (11 th day post-infection (dpi). (B) Neutrophil number (Ly6G + CD11b + ) and (C) Ncf2 gene expression in the lungs. (D) KC (CXCL-1) and (E) MIP-2 (CXCL-2) in the sera of mice with ALI/ARDS (red) and HP (black) on the 3 rd and 5 th dpi. Number of neutrophils (Ly6G + CD11b + ) producing (F) reactive oxygen species (DHR + ) in the lungs or producing myeloperoxidase (MPO) in the (G) lungs and (H) bronchoalveolar lavage fluid (BALF) of mice with ALI/ARDS compared with those from mice with HP. (I e J) Leukocytes in the BALF of mice. Arrowheads indicate neutrophils (400x, scale bar 25 μm). Data for three grouped experiments are expressed as the mean ± SEM. The data from figures B, C, F, G, H, I and J were collected on the 7 th dpi. (Mann-Whitney test and Kruskal-Wallis test; (n = 5); ALI/ARDS: n = 12–20, HP: n = 10–15; * p

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Mouse Assay, Infection, Staining, Expressing, MANN-WHITNEY

    Plasmodium falciparum and Plasmodium berghei induce neutrophil extracellular trap (NET) formation. (A) Immunofluorescence of a human neutrophil culture stimulated with PMA (positive control) and P . falciparum (iRBCs) stained for neutrophil elastase (green), histone (red) and DNA (blue). Overlapping colors indicate filamentous structures (NETs) (400x, scale bar 25 μm). (B) Immunofluorescence of a DBA/2 mice neutrophil culture stimulated with PMA (positive control), red blood cells (RBCs), P . berghei lysate and P . berghei ANKA (iRBCs) stained for myeloperoxidase (green), histone (red), and DNA (blue) with overlapping pictures (1000x, scale bar 20 μm).

    Journal: PLoS Pathogens

    Article Title: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

    doi: 10.1371/journal.ppat.1006054

    Figure Lengend Snippet: Plasmodium falciparum and Plasmodium berghei induce neutrophil extracellular trap (NET) formation. (A) Immunofluorescence of a human neutrophil culture stimulated with PMA (positive control) and P . falciparum (iRBCs) stained for neutrophil elastase (green), histone (red) and DNA (blue). Overlapping colors indicate filamentous structures (NETs) (400x, scale bar 25 μm). (B) Immunofluorescence of a DBA/2 mice neutrophil culture stimulated with PMA (positive control), red blood cells (RBCs), P . berghei lysate and P . berghei ANKA (iRBCs) stained for myeloperoxidase (green), histone (red), and DNA (blue) with overlapping pictures (1000x, scale bar 20 μm).

    Article Snippet: Neutrophils were stimulated with synchronized P . berghei- iRBCs, P . berghei lysate, non-infected red blood cells (RBCs) and PMA (50 nM; Sigma-Aldrich, USA) for 3 hours at 37°C in a humidified atmosphere containing 5% CO2 .

    Techniques: Immunofluorescence, Positive Control, Staining, Mouse Assay

    DON induces transient PKR activation in HeLa-based cell-free system. ( A ) DON (100, 250, 1000 ng/mL), poly (IC) (100 ng/mL) and/or PKR inhibitors, 2-AP (2 mM) and C16 (2 µM), were added to a cell-free system comprised of HeLa-based cell-derived, translationally active cell-free system containing ribosomes and ATP but devoid of cell membrane, nuclei, mitochondria, DNA and mRNA. After incubation for 20 min at 30 °C, Western analysis was conducted with PKR and p-PKR antibodies; ( B ) HeLa-based cell-free assay mixtures were incubated with DON (250 ng/mL) for indicated time intervals and subjected to Western blotting with PKR, p-PKR and eIF2α, p -eIF2α antibodies. Data are representative of at least three replicate experiments.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: DON induces transient PKR activation in HeLa-based cell-free system. ( A ) DON (100, 250, 1000 ng/mL), poly (IC) (100 ng/mL) and/or PKR inhibitors, 2-AP (2 mM) and C16 (2 µM), were added to a cell-free system comprised of HeLa-based cell-derived, translationally active cell-free system containing ribosomes and ATP but devoid of cell membrane, nuclei, mitochondria, DNA and mRNA. After incubation for 20 min at 30 °C, Western analysis was conducted with PKR and p-PKR antibodies; ( B ) HeLa-based cell-free assay mixtures were incubated with DON (250 ng/mL) for indicated time intervals and subjected to Western blotting with PKR, p-PKR and eIF2α, p -eIF2α antibodies. Data are representative of at least three replicate experiments.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Activation Assay, Derivative Assay, Incubation, Western Blot, Cell-Free Assay

    Trichothecene deoxynivalenol (DON) induces protein kinase (PKR)-dependent ribotoxic stress response in HeLa cells. Kinetics of DON-induced phosphorylation of ( A ) p38; and ( B ) c-jun N-terminal kinase(JNK) HeLa cells were treated with 500 ng/mL of DON for indicated time intervals and then p38 and JNK phosphorylation was determined by Western analysis; ( C ) PKR inhibitor 2-aminopurine (2-AP) suppresses DON-induced p38 and JNK phosphorylation. HeLa cells were pretreated with PKR inhibitor 2-AP at indicated concentrations for 1 h, exposed to DON (500 ng/mL) for 15 min and then p38 and JNK phosphorylation was determined by Western analysis. Data are representative of at least two replicate experiments.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Trichothecene deoxynivalenol (DON) induces protein kinase (PKR)-dependent ribotoxic stress response in HeLa cells. Kinetics of DON-induced phosphorylation of ( A ) p38; and ( B ) c-jun N-terminal kinase(JNK) HeLa cells were treated with 500 ng/mL of DON for indicated time intervals and then p38 and JNK phosphorylation was determined by Western analysis; ( C ) PKR inhibitor 2-aminopurine (2-AP) suppresses DON-induced p38 and JNK phosphorylation. HeLa cells were pretreated with PKR inhibitor 2-AP at indicated concentrations for 1 h, exposed to DON (500 ng/mL) for 15 min and then p38 and JNK phosphorylation was determined by Western analysis. Data are representative of at least two replicate experiments.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Western Blot

    Ribotoxins do not affect rRNA integrity prior to PKR activation in HeLa-based cell-free assay mixtures. HeLa-based cell-free assay mixtures were incubated with vehicle, DON, anisomycin (20 ng/mL) or ricin (20 ng/mL) for 30 min at 30 °C and then subjected to RNA purification and capillary electrophoresis. Data are representative of at least three replicate experiments.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Ribotoxins do not affect rRNA integrity prior to PKR activation in HeLa-based cell-free assay mixtures. HeLa-based cell-free assay mixtures were incubated with vehicle, DON, anisomycin (20 ng/mL) or ricin (20 ng/mL) for 30 min at 30 °C and then subjected to RNA purification and capillary electrophoresis. Data are representative of at least three replicate experiments.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Activation Assay, Cell-Free Assay, Incubation, Purification, Electrophoresis

    Ricin induces transient PKR activation in HeLa cell-free system. Experiment conditions were as described in Figure 4 legend except that ricin was used instead of anisomycin.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Ricin induces transient PKR activation in HeLa cell-free system. Experiment conditions were as described in Figure 4 legend except that ricin was used instead of anisomycin.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Activation Assay

    Distribution of RNA-IP-identified PKR-associated sequences in HeLa-based cell-free assay mixtures. HeLa lysate assay mixtures were incubated with or without DON (250 ng/mL) at 30 °C for 20 min and subjected to RNA-IP using PKR-specific antibody in four independent experiments. Following purification, cloning and sequencing of immunoprecipitated PKR-associated rRNAs, sequences were aligned to human 18S (NCBI Reference Sequence: NR-003286.2) and 28S (NCBI Reference Sequence: NR_03287.2) ribosomal RNA genes and their relative sizes and locations depicted above. The upper (solid) and lower (dotted) lines indicate the PKR-associated sequences identified from DON-treated and control, respectively. Integers in parentheses denote the number of clones recovered that were recovered in designated region.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Distribution of RNA-IP-identified PKR-associated sequences in HeLa-based cell-free assay mixtures. HeLa lysate assay mixtures were incubated with or without DON (250 ng/mL) at 30 °C for 20 min and subjected to RNA-IP using PKR-specific antibody in four independent experiments. Following purification, cloning and sequencing of immunoprecipitated PKR-associated rRNAs, sequences were aligned to human 18S (NCBI Reference Sequence: NR-003286.2) and 28S (NCBI Reference Sequence: NR_03287.2) ribosomal RNA genes and their relative sizes and locations depicted above. The upper (solid) and lower (dotted) lines indicate the PKR-associated sequences identified from DON-treated and control, respectively. Integers in parentheses denote the number of clones recovered that were recovered in designated region.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Cell-Free Assay, Incubation, Purification, Clone Assay, Sequencing, Immunoprecipitation

    DON does not alter PKR binding to rRNA in HeLa-based cell-free assay mixtures. ( A ) Sequences and sizes of 32P labeled probes for 18S-U1 28S-U2 and 28S-C1; ( B ) Radiolabeled probes were incubated with immunoprecipitated RNAs from control and DON-treated HeLa extracts, subjected to RNase treatment, separated by urea-PAGE and analyzed by autoradiography.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: DON does not alter PKR binding to rRNA in HeLa-based cell-free assay mixtures. ( A ) Sequences and sizes of 32P labeled probes for 18S-U1 28S-U2 and 28S-C1; ( B ) Radiolabeled probes were incubated with immunoprecipitated RNAs from control and DON-treated HeLa extracts, subjected to RNase treatment, separated by urea-PAGE and analyzed by autoradiography.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Binding Assay, Cell-Free Assay, Labeling, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Multiple PKR molecules associate with 40S, 60S, 80S and polysomes in naïve HeLa cells. Stoichiometry was assessed by density gradient fractionation of ribosomes from naive HeLa cell lysate in conjunction with quantitative Western blotting using the Li-Cor Odyssey Infrared Imaging System. M indicates molecular weight markers. ND indicates not determined; Results are representative of three identical.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Multiple PKR molecules associate with 40S, 60S, 80S and polysomes in naïve HeLa cells. Stoichiometry was assessed by density gradient fractionation of ribosomes from naive HeLa cell lysate in conjunction with quantitative Western blotting using the Li-Cor Odyssey Infrared Imaging System. M indicates molecular weight markers. ND indicates not determined; Results are representative of three identical.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Fractionation, Western Blot, Imaging, Molecular Weight

    Predicted secondary structures of rRNAs recovered by PKR-specific RNA-IP from HeLa-based cell-free assay mixtures contain significant double-stranded regions. Representative PKR-bound rRNA sequences (( A ) 18S-U1; ( C ) 28S-U2; and ( E ) 28S-C1) were mapped and aligned to human 18S and 28S rRNAs and then depicted with PseudoViewer. Dashed lines indicate regions of dsrRNA available for interaction with PKR DRBD based on predicted secondary structure of the fragment. In addition, secondary structures of the isolated PKR-associated rRNA fragments (( B ) 18S-U1; ( D ) 28S-U2; and ( F ) 28S-C1) were predicted to confirm double-stranded regions using PknotsRG with minimum free energy and then depicted with PseudoViewer.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Predicted secondary structures of rRNAs recovered by PKR-specific RNA-IP from HeLa-based cell-free assay mixtures contain significant double-stranded regions. Representative PKR-bound rRNA sequences (( A ) 18S-U1; ( C ) 28S-U2; and ( E ) 28S-C1) were mapped and aligned to human 18S and 28S rRNAs and then depicted with PseudoViewer. Dashed lines indicate regions of dsrRNA available for interaction with PKR DRBD based on predicted secondary structure of the fragment. In addition, secondary structures of the isolated PKR-associated rRNA fragments (( B ) 18S-U1; ( D ) 28S-U2; and ( F ) 28S-C1) were predicted to confirm double-stranded regions using PknotsRG with minimum free energy and then depicted with PseudoViewer.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Cell-Free Assay, Isolation

    Anisomycin induces transient PKR activation in HeLa cell-free system. ( A ) Anisomycin (10 to 50 ng/mL), poly (IC) (100 ng/mL) and/or PKR inhibitors, 2-AP (2 mM) and C16 (2 µM), were added to HeLa-based cell-free assay mixtures. After incubation for 20 min at 30 °C, Western analysis was conducted with PKR and p-PKR antibodies; ( B ) HeLa-based cell-free assay mixtures were incubated with anisomycin (20 ng/mL) for indicated time intervals and subjected to Western blotting analysis with PKR, p-PKR and eIF2α, p-eIF2α antibodies. Data are representative of at least three replicate experiments.

    Journal: Toxins

    Article Title: Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    doi: 10.3390/toxins6123406

    Figure Lengend Snippet: Anisomycin induces transient PKR activation in HeLa cell-free system. ( A ) Anisomycin (10 to 50 ng/mL), poly (IC) (100 ng/mL) and/or PKR inhibitors, 2-AP (2 mM) and C16 (2 µM), were added to HeLa-based cell-free assay mixtures. After incubation for 20 min at 30 °C, Western analysis was conducted with PKR and p-PKR antibodies; ( B ) HeLa-based cell-free assay mixtures were incubated with anisomycin (20 ng/mL) for indicated time intervals and subjected to Western blotting analysis with PKR, p-PKR and eIF2α, p-eIF2α antibodies. Data are representative of at least three replicate experiments.

    Article Snippet: The effects of DON, anisomycin (Sigma-Aldrich) and ricin (Vector, Burlingame, CA, USA) on phosphorylation of PKR and its substrate eIF2α were determined under cell-free conditions employing a translationally-active HeLa cell lysate (Thermo Scientific, Wilmington, DE, USA).

    Techniques: Activation Assay, Cell-Free Assay, Incubation, Western Blot

    Clonotypic distribution of T cells comparing EBV − HIV + versus EBV + HIV + patients under EBV-stimulated conditions. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro with EBV lysate. The distribution of CD4 + (A) and CD8 + (B) T cells positive for TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Clonotypic distribution of T cells comparing EBV − HIV + versus EBV + HIV + patients under EBV-stimulated conditions. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro with EBV lysate. The distribution of CD4 + (A) and CD8 + (B) T cells positive for TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Cell Culture, In Vitro, Flow Cytometry, Cytometry, Whisker Assay, MANN-WHITNEY

    Clonotypic distribution of T cells from EBV + HIV + patients. Peripheral blood samples of HIV + patients were cultured in vitro at basal (without EBV) conditions. The distribution of CD4 + and CD8 + T cells positive for some of 24 TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for some individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Clonotypic distribution of T cells from EBV + HIV + patients. Peripheral blood samples of HIV + patients were cultured in vitro at basal (without EBV) conditions. The distribution of CD4 + and CD8 + T cells positive for some of 24 TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for some individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Cell Culture, In Vitro, Flow Cytometry, Cytometry, Whisker Assay, MANN-WHITNEY

    Functional EBV-specific T cell response in EBV − HIV + vs. EBV + HIV + patients. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro with EBV lysate. Counts of mono- and multifunctional (TNF-α + , IFN-γ and/or IL-2) CD4 + (A) and CD8 + (B) T cells were analyzed by flow cytometry. Net (EBV minus basal) counts are shown. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Functional EBV-specific T cell response in EBV − HIV + vs. EBV + HIV + patients. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro with EBV lysate. Counts of mono- and multifunctional (TNF-α + , IFN-γ and/or IL-2) CD4 + (A) and CD8 + (B) T cells were analyzed by flow cytometry. Net (EBV minus basal) counts are shown. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Functional Assay, Cell Culture, In Vitro, Flow Cytometry, Cytometry, MANN-WHITNEY

    Cytokines synthesized in response to EBV by HIV + patients. Peripheral blood samples of HIV + patients at different stages of disease and healthy controls were cultured in vitro at basal (without EBV) and EBV-stimulated conditions without BFA. Supernatants were collected to measure the concentration (pg/mL) of cytokines by CBA and flow cytometry. The levels of IFN-γ (A) , IL-2 (B) , IL-4 (C) , IL-6 (D) , IL-10 (E) and TNF-α ( F ), are shown, respectively. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups of individuals. Wilcoxon signed-rank Test was used for comparison between basal and EBV conditions. Dotted lines correspond to the limit of detection for every cytokine.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Cytokines synthesized in response to EBV by HIV + patients. Peripheral blood samples of HIV + patients at different stages of disease and healthy controls were cultured in vitro at basal (without EBV) and EBV-stimulated conditions without BFA. Supernatants were collected to measure the concentration (pg/mL) of cytokines by CBA and flow cytometry. The levels of IFN-γ (A) , IL-2 (B) , IL-4 (C) , IL-6 (D) , IL-10 (E) and TNF-α ( F ), are shown, respectively. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups of individuals. Wilcoxon signed-rank Test was used for comparison between basal and EBV conditions. Dotted lines correspond to the limit of detection for every cytokine.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Synthesized, Cell Culture, In Vitro, Concentration Assay, Crocin Bleaching Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Clonotypic distribution of T cells from HIV + patients at basal conditions. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro at basal (without EBV) conditions. The distribution of CD4 + (A) and CD8 + (B) T cells positive for any of 24 TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Clonotypic distribution of T cells from HIV + patients at basal conditions. Peripheral blood samples of HIV + patients and healthy controls were cultured in vitro at basal (without EBV) conditions. The distribution of CD4 + (A) and CD8 + (B) T cells positive for any of 24 TCR-Vβ families was analyzed with specific mAbs and flow cytometry. Box and whisker plots show range, median, and interquartile range of percentage of T cells positive for individual Vβ families. Mann-Whitney U -test was used for comparisons between groups.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Cell Culture, In Vitro, Flow Cytometry, Cytometry, Whisker Assay, MANN-WHITNEY

    Cytokine response to a polyclonal stimulus in EBV + HIV + patients. Peripheral blood samples of HIV + patients were cultured in vitro at basal (without stimulus) or polyclonal (PMA + ionomycin)-stimulated conditions with BFA. Monofunctional and multifunctional (TNF-α + , IFN-γ + and/ or IL-2 + ) T cells were characterized using mAbs and flow cytometry. The results for CD8 + T cells are shown (A) . Parallel cultures were made without BFA for collecting supernatants and measuring the concentration of soluble cytokines by CBA and flow cytometry. Supernatant IL-6 at basal condition (B) and supernatant TNF-α at polyclonally stimulated condition (C) . Bold lines represent median values. Dotted lines correspond to the limit of detection for each cytokine. Mann-Whitney U -test was used for comparisons between groups of individuals.

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Cytokine response to a polyclonal stimulus in EBV + HIV + patients. Peripheral blood samples of HIV + patients were cultured in vitro at basal (without stimulus) or polyclonal (PMA + ionomycin)-stimulated conditions with BFA. Monofunctional and multifunctional (TNF-α + , IFN-γ + and/ or IL-2 + ) T cells were characterized using mAbs and flow cytometry. The results for CD8 + T cells are shown (A) . Parallel cultures were made without BFA for collecting supernatants and measuring the concentration of soluble cytokines by CBA and flow cytometry. Supernatant IL-6 at basal condition (B) and supernatant TNF-α at polyclonally stimulated condition (C) . Bold lines represent median values. Dotted lines correspond to the limit of detection for each cytokine. Mann-Whitney U -test was used for comparisons between groups of individuals.

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Cell Culture, In Vitro, Flow Cytometry, Cytometry, Concentration Assay, Crocin Bleaching Assay, MANN-WHITNEY

    Functional EBV-specific T cell response in HIV + patients. Peripheral blood samples of HIV + patients at different stages of disease and healthy controls were cultured in vitro with EBV lysate. Counts of mono- and multifunctional (TNF-α + , IFN-γ and/or IL-2) CD4 + (A) and CD8 + (B) T cells were analyzed by flow cytometry. Net (EBV minus basal) counts are shown. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups. (C) CD4 + and CD8 + T cells that coexpress intracellular cytokines after stimulation with EBV are shown. The colors in the pie charts depict the coexpression of the cytokines: one (dark blue), two (light blue, red and light green) and three (dark green). The p values of the permutation test in the coexpression analysis are shown (* p

    Journal: Frontiers in Immunology

    Article Title: Loss of T-Cell Multifunctionality and TCR-Vβ Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients

    doi: 10.3389/fimmu.2018.02291

    Figure Lengend Snippet: Functional EBV-specific T cell response in HIV + patients. Peripheral blood samples of HIV + patients at different stages of disease and healthy controls were cultured in vitro with EBV lysate. Counts of mono- and multifunctional (TNF-α + , IFN-γ and/or IL-2) CD4 + (A) and CD8 + (B) T cells were analyzed by flow cytometry. Net (EBV minus basal) counts are shown. Bold lines represent median values. Mann-Whitney U -test was used for comparisons between groups. (C) CD4 + and CD8 + T cells that coexpress intracellular cytokines after stimulation with EBV are shown. The colors in the pie charts depict the coexpression of the cytokines: one (dark blue), two (light blue, red and light green) and three (dark green). The p values of the permutation test in the coexpression analysis are shown (* p

    Article Snippet: Similar results in HIV+ patients with a high viral load found a decrease in IFN-γ+ /IL-2+ CD4+ T cell counts in response to an EBV lysate, together with high expression of activating molecules ( ).

    Techniques: Functional Assay, Cell Culture, In Vitro, Flow Cytometry, Cytometry, MANN-WHITNEY

    IKK-inhibiting viral degradation of Foxj1 and CSF/brain barrier disruption. a IHC staining of primary EC cultures without (Ctrl) or with indicated treatment conditions, labeled with Foxj1 and VP16 antibodies, and DAPI. Cultures were treated with viruses for 16 h. Quantifications showing Foxj1 + cells per total DAPI nuclei in treatment conditions as percentage of Ctrl condition in each experiment (read dashed line). * P

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: IKK-inhibiting viral degradation of Foxj1 and CSF/brain barrier disruption. a IHC staining of primary EC cultures without (Ctrl) or with indicated treatment conditions, labeled with Foxj1 and VP16 antibodies, and DAPI. Cultures were treated with viruses for 16 h. Quantifications showing Foxj1 + cells per total DAPI nuclei in treatment conditions as percentage of Ctrl condition in each experiment (read dashed line). * P

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Immunohistochemistry, Staining, Labeling

    Ex vivo live imaging of mature ependymal cell transformation. a foxj1 CreERt2/+ ; R26R-tdT mice tamoxifen induction and wholemount imaging (dashed red line) schedule. b Representative time-lapsed images from foxj1 CreERt2/+ ; R26R-tdT ependymal wholemount sample cultured in high serum proliferation media (Prolif. media) condition. Note the cellular transformation of tdTomato + ECs over time. c Representative close-up view of individual tdTomato + EC in Prolif. media condition, showing morphological transformation over time, with radial glial-like processes (arrowheads) emanating from cell body (arrows). d Quantification of transformed tdTomato + EC numbers for control (Ctrl) and Prolif. media conditions. * P

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: Ex vivo live imaging of mature ependymal cell transformation. a foxj1 CreERt2/+ ; R26R-tdT mice tamoxifen induction and wholemount imaging (dashed red line) schedule. b Representative time-lapsed images from foxj1 CreERt2/+ ; R26R-tdT ependymal wholemount sample cultured in high serum proliferation media (Prolif. media) condition. Note the cellular transformation of tdTomato + ECs over time. c Representative close-up view of individual tdTomato + EC in Prolif. media condition, showing morphological transformation over time, with radial glial-like processes (arrowheads) emanating from cell body (arrows). d Quantification of transformed tdTomato + EC numbers for control (Ctrl) and Prolif. media conditions. * P

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Ex Vivo, Imaging, Transformation Assay, Mouse Assay, Cell Culture

    De-differentiation, cell division, and re-differentiation of mature ECs. a Representative time-lapsed images from P14 foxj1 CreERt2/+ ; R26R-tdT ependymal wholemount sample, tamoxifen-induced at P7, cultured in proliferation media for indicated hours. Note the lineage-traced tdTomato + cell (arrows) undergoing cell division (tracked by blue dots). Scale bar: 20 µm. b IHC staining of ependymal wholemount from P14 foxj1 CreERt2/+ ; R26R-tdT animal, P7 tamoxifen-induced, cultured in proliferation media condition for 72 h, and labeled with RFP, EdU (pulsed at 36 h), and DAPI, showing EdU incorporation in lineage-traced tdTomato + cells (arrows). Scale bar: 20 µm. c Quantification = % of lineage-traced tdTomato + cells containing multicilia (marked by A-tub). * P

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: De-differentiation, cell division, and re-differentiation of mature ECs. a Representative time-lapsed images from P14 foxj1 CreERt2/+ ; R26R-tdT ependymal wholemount sample, tamoxifen-induced at P7, cultured in proliferation media for indicated hours. Note the lineage-traced tdTomato + cell (arrows) undergoing cell division (tracked by blue dots). Scale bar: 20 µm. b IHC staining of ependymal wholemount from P14 foxj1 CreERt2/+ ; R26R-tdT animal, P7 tamoxifen-induced, cultured in proliferation media condition for 72 h, and labeled with RFP, EdU (pulsed at 36 h), and DAPI, showing EdU incorporation in lineage-traced tdTomato + cells (arrows). Scale bar: 20 µm. c Quantification = % of lineage-traced tdTomato + cells containing multicilia (marked by A-tub). * P

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Cell Culture, Immunohistochemistry, Staining, Labeling

    Effects of MLN4924 on ependymal Foxj1 transcription factor expression. a Representative IHC staining of control (Ctrl) and MLN4924-treated primary EC cultures, labeled with acetylated tubulin (A-tub) antibody and DAPI. Scale bar: 50 µm. b Quantification of multiciliated cell numbers as a fraction of DAPI + nuclei. * P

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: Effects of MLN4924 on ependymal Foxj1 transcription factor expression. a Representative IHC staining of control (Ctrl) and MLN4924-treated primary EC cultures, labeled with acetylated tubulin (A-tub) antibody and DAPI. Scale bar: 50 µm. b Quantification of multiciliated cell numbers as a fraction of DAPI + nuclei. * P

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Expressing, Immunohistochemistry, Staining, Labeling

    Ependymal Foxj1 protein instability and turnover. a , c , d , h , k Western blot (WB) analyses of protein lysates from primary EC cultures. a Foxj1 protein levels before and after treatment with cycloheximide (CHX) for indicated lengths of time. Actin = loading control. b IHC staining of EC cultures treated with CHX for the indicated lengths of time, labeled with Foxj1 antibody. Foxj1 is lost at a similar timescale as observed in WBs. c Foxj1 protein levels in untreated controls, CHX-treated for 3 h, or co-treated with CHX + MG132 for 3 h. Actin = loading control. d Foxj1 protein levels in untreated controls, CHX-treated, or co-treated with CHX + MLN4924 for the indicated lengths of time. Actin = loading control. e Immunoprecipitation (IP) of Foxj1 from EC cultures followed by WB probing for Foxj1 to reveal ubiquitin-induced laddering of Foxj1 protein. Lane 1 = input, lane 2 = IP Foxj1, lane 3 = IP of Foxj1 after MG132 treatment for 6 h. f IP of ubiquitin from EC cultures followed by WB probing for Foxj1. Lane 1 = input, lane 2 = IP ubiquitin, lane 3 = IP ubiquitin following MG132 treatment for 6 h. g IP of Foxj1 from EC cultures followed by WB probing for ubiquitin. Lane 1 = input, lane 2 = IP of Foxj1, lane 3 = IP of Foxj1 following MG132 treatment for 6 h. h Cultures treated with DMSO control (Ctrl) or SMER3, probed for Foxj1 and Actin. i Representative IHC staining of primary EC cultures without (Ctrl) or with SMER3 treatment, labeled with Foxj1 antibody and DAPI. Note the increased Foxj1 IHC signal in SMER3-treated EC cultures. j IHC staining of differentiated EC cultures labeled with Fbxw5, Foxj1 antibodies, and DAPI. k Cultures harboring scrambled control (Scr) shRNA or Fbxw5 shRNA, treated with CHX for indicated lengths of time, and probed for Foxj1 and Actin. l IP of Foxj1 from HEK293 cells either co-transfected with GFP and Foxj1-Myc expression constructs, or Fbxw5 and Foxj1-Myc constructs. WBs were probed with GFP or Fbxw5 antibodies. Lane 1 = inputs, Lane 2 = IP of Foxj1, Lane 3 = IP of Foxj1 following 6 h MG132 treatment. m IP of Foxj1 from HEK293 cell lysates following co-transfection with expression constructs as indicated (+), probed with anti-HA antibody to detect HA-ubiquitin. Scale bars: 20 µm

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: Ependymal Foxj1 protein instability and turnover. a , c , d , h , k Western blot (WB) analyses of protein lysates from primary EC cultures. a Foxj1 protein levels before and after treatment with cycloheximide (CHX) for indicated lengths of time. Actin = loading control. b IHC staining of EC cultures treated with CHX for the indicated lengths of time, labeled with Foxj1 antibody. Foxj1 is lost at a similar timescale as observed in WBs. c Foxj1 protein levels in untreated controls, CHX-treated for 3 h, or co-treated with CHX + MG132 for 3 h. Actin = loading control. d Foxj1 protein levels in untreated controls, CHX-treated, or co-treated with CHX + MLN4924 for the indicated lengths of time. Actin = loading control. e Immunoprecipitation (IP) of Foxj1 from EC cultures followed by WB probing for Foxj1 to reveal ubiquitin-induced laddering of Foxj1 protein. Lane 1 = input, lane 2 = IP Foxj1, lane 3 = IP of Foxj1 after MG132 treatment for 6 h. f IP of ubiquitin from EC cultures followed by WB probing for Foxj1. Lane 1 = input, lane 2 = IP ubiquitin, lane 3 = IP ubiquitin following MG132 treatment for 6 h. g IP of Foxj1 from EC cultures followed by WB probing for ubiquitin. Lane 1 = input, lane 2 = IP of Foxj1, lane 3 = IP of Foxj1 following MG132 treatment for 6 h. h Cultures treated with DMSO control (Ctrl) or SMER3, probed for Foxj1 and Actin. i Representative IHC staining of primary EC cultures without (Ctrl) or with SMER3 treatment, labeled with Foxj1 antibody and DAPI. Note the increased Foxj1 IHC signal in SMER3-treated EC cultures. j IHC staining of differentiated EC cultures labeled with Fbxw5, Foxj1 antibodies, and DAPI. k Cultures harboring scrambled control (Scr) shRNA or Fbxw5 shRNA, treated with CHX for indicated lengths of time, and probed for Foxj1 and Actin. l IP of Foxj1 from HEK293 cells either co-transfected with GFP and Foxj1-Myc expression constructs, or Fbxw5 and Foxj1-Myc constructs. WBs were probed with GFP or Fbxw5 antibodies. Lane 1 = inputs, Lane 2 = IP of Foxj1, Lane 3 = IP of Foxj1 following 6 h MG132 treatment. m IP of Foxj1 from HEK293 cell lysates following co-transfection with expression constructs as indicated (+), probed with anti-HA antibody to detect HA-ubiquitin. Scale bars: 20 µm

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Western Blot, Immunohistochemistry, Staining, Labeling, Immunoprecipitation, shRNA, Transfection, Expressing, Construct, Cotransfection, Hemagglutination Assay

    IKK function in Foxj1 stability. a Transcriptome heatmap (FDR ≤ 0.05) comparing EC cultures without (Ctrl) or with MLN4924 treatment, Z -score normalized. Samples/genes are clustered by correlation distance with complete linkage. b Gene enrichment plot of IKK/NF-κB cascade identified from gene set enrichment analysis (GSEA), using gene ontology (GO) database. Genes ranked on significance and direction of change ( t -statistic). List of genes enriched within pathway (listed beneath) is presented with correlated heatmap varying in color from black to red. c Protein interaction network from identified genes within IKK/NF-κB cascade, using STRING functional protein association network. Connecting line thickness corresponds to network association confidence. d , f , g , m Western blot (WB) analyses of protein lysates from primary EC cultures. d Cultures treated with IMD-0354 for the indicated lengths of times, and probed for Foxj1, Actin (loading control). e Representative IHC staining of primary EC cultures without (Ctrl) or with addition of indicated compounds and combinations for 6 h, labeled with Foxj1 antibody and DAPI. Scale bar: 20 µm. f Cultures treated for 6 h with IMD-0354 alone or IMD-0354 + MG132, and probed for Foxj1, Actin. g Cultures untreated or treated for 6 h with MLN4924 or IMD-0354 + MLN4924, and probed for Foxj1, Actin. h Modified SDS-page gel protocol separating closely migrating molecular weight bands followed by WB for Foxj1, Actin. ECs were treated with IMD-0354 for indicated lengths of time. i , j , l Western blot (WB) analyses of protein lysates from HEK293 cells. i Cultures transfected with indicated expression constructs, and probed with anti-Foxj1 and anti-Flag (IKK2) antibodies. IKK2-Flag levels are used as expression controls. Note the formation of a Foxj1 doublet in the presence of IKK2(Wt) but not the IKK2(Mut) (kinase dead) conditions. j Cultures co-transfected with the indicated expression constructs and drug treatments, probed with anti-Foxj1, anti-Flag (IKK2) antibodies. IKK2-Flag levels = expression controls (lower lane). k IKK2 phosphorylation motif, comparing IκBα, Foxj1 proteins from several species. Alignment of target serines highlighted in blue. l Cultures co-transfected with expression constructs as indicated (+), probed with anti-Foxj1 antibody. m Cultures expressing indicated Foxj1 constructs, probed with anti-Myc antibody. Comparison of cultures untreated vs. treated with CHX for 6 h, showing enhanced stability of S32D/S35D (S/D)-Foxj1-Myc mutant protein (not S/A-Foxj1-Myc). n IP from HEK293 cell lysates co-transfected with expression constructs as indicated (+), probed with Myc(Foxj1) and Flag(IKK2) antibodies. IKK2-Flag and Foxj1-Myc levels are used as expression controls (lower lanes)

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: IKK function in Foxj1 stability. a Transcriptome heatmap (FDR ≤ 0.05) comparing EC cultures without (Ctrl) or with MLN4924 treatment, Z -score normalized. Samples/genes are clustered by correlation distance with complete linkage. b Gene enrichment plot of IKK/NF-κB cascade identified from gene set enrichment analysis (GSEA), using gene ontology (GO) database. Genes ranked on significance and direction of change ( t -statistic). List of genes enriched within pathway (listed beneath) is presented with correlated heatmap varying in color from black to red. c Protein interaction network from identified genes within IKK/NF-κB cascade, using STRING functional protein association network. Connecting line thickness corresponds to network association confidence. d , f , g , m Western blot (WB) analyses of protein lysates from primary EC cultures. d Cultures treated with IMD-0354 for the indicated lengths of times, and probed for Foxj1, Actin (loading control). e Representative IHC staining of primary EC cultures without (Ctrl) or with addition of indicated compounds and combinations for 6 h, labeled with Foxj1 antibody and DAPI. Scale bar: 20 µm. f Cultures treated for 6 h with IMD-0354 alone or IMD-0354 + MG132, and probed for Foxj1, Actin. g Cultures untreated or treated for 6 h with MLN4924 or IMD-0354 + MLN4924, and probed for Foxj1, Actin. h Modified SDS-page gel protocol separating closely migrating molecular weight bands followed by WB for Foxj1, Actin. ECs were treated with IMD-0354 for indicated lengths of time. i , j , l Western blot (WB) analyses of protein lysates from HEK293 cells. i Cultures transfected with indicated expression constructs, and probed with anti-Foxj1 and anti-Flag (IKK2) antibodies. IKK2-Flag levels are used as expression controls. Note the formation of a Foxj1 doublet in the presence of IKK2(Wt) but not the IKK2(Mut) (kinase dead) conditions. j Cultures co-transfected with the indicated expression constructs and drug treatments, probed with anti-Foxj1, anti-Flag (IKK2) antibodies. IKK2-Flag levels = expression controls (lower lane). k IKK2 phosphorylation motif, comparing IκBα, Foxj1 proteins from several species. Alignment of target serines highlighted in blue. l Cultures co-transfected with expression constructs as indicated (+), probed with anti-Foxj1 antibody. m Cultures expressing indicated Foxj1 constructs, probed with anti-Myc antibody. Comparison of cultures untreated vs. treated with CHX for 6 h, showing enhanced stability of S32D/S35D (S/D)-Foxj1-Myc mutant protein (not S/A-Foxj1-Myc). n IP from HEK293 cell lysates co-transfected with expression constructs as indicated (+), probed with Myc(Foxj1) and Flag(IKK2) antibodies. IKK2-Flag and Foxj1-Myc levels are used as expression controls (lower lanes)

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Functional Assay, Western Blot, Radial Immuno Diffusion, Immunohistochemistry, Staining, Labeling, Modification, SDS Page, Molecular Weight, Transfection, Expressing, Construct, Mutagenesis

    Inducible deletion of Foxj1 from mature ECs. a Schematic representation of foxj1 CreERt2/+ gene targeting strategy, inserting CreER t2 into ATG site encoding Foxj1. N = NotI, E = EcoRI. Recombined allele was crossed to Rosa26-fl p , removing neomycin selection cassette ( neo ) for proper CreER t2 recombinase expression from foxj1 gene locus. b – g All experimental animals were tamoxifen induced at P14, samples harvested at P28. b Representative IHC staining of ependymal wholemount from foxj1 CreERt2/+ ; R26R-tdT animal, labeled with acetylated tubulin (A-tub) and RFP antibodies. Dashed circles indicate multicilia bundles (upper panel) from individual tdTomato + ECs. Quantification = % of tdTomato + cells showing multicilia, from four animals. c Representative IHC staining of brain sections from foxj1 CreERt2/+ ; R26R-tdT and foxj1 CreERt2/Flox ; R26R-tdT animals, labeled with A-tub and RFP antibodies, and DAPI, showing loss of multicilia in lineage-traced Foxj1 mutant tdTomato + ECs. Quantification = % of tdTomato + cells containing multicilia. * P

    Journal: Nature Communications

    Article Title: Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus

    doi: 10.1038/s41467-018-03812-w

    Figure Lengend Snippet: Inducible deletion of Foxj1 from mature ECs. a Schematic representation of foxj1 CreERt2/+ gene targeting strategy, inserting CreER t2 into ATG site encoding Foxj1. N = NotI, E = EcoRI. Recombined allele was crossed to Rosa26-fl p , removing neomycin selection cassette ( neo ) for proper CreER t2 recombinase expression from foxj1 gene locus. b – g All experimental animals were tamoxifen induced at P14, samples harvested at P28. b Representative IHC staining of ependymal wholemount from foxj1 CreERt2/+ ; R26R-tdT animal, labeled with acetylated tubulin (A-tub) and RFP antibodies. Dashed circles indicate multicilia bundles (upper panel) from individual tdTomato + ECs. Quantification = % of tdTomato + cells showing multicilia, from four animals. c Representative IHC staining of brain sections from foxj1 CreERt2/+ ; R26R-tdT and foxj1 CreERt2/Flox ; R26R-tdT animals, labeled with A-tub and RFP antibodies, and DAPI, showing loss of multicilia in lineage-traced Foxj1 mutant tdTomato + ECs. Quantification = % of tdTomato + cells containing multicilia. * P

    Article Snippet: To separate the Foxj1 doublet lysates were allowed to migrate through premade 4–20% gradient Tris-Glycine gels (Bio-Rad) for 1.5 h at 170 volts.

    Techniques: Selection, Expressing, Immunohistochemistry, Staining, Labeling, Mutagenesis

    Schematic representation of Ar/NH 3 plasma-treated MNBs and their binding to anti-virus antibody. Notes: Graphite-encapsulated MNBs were treated with ammonia plasma and modified with amino groups. SPDP was reacted with the amino group of modified MNBs (NH 2 -beads) at pH 7–8. Anti-rotavirus antibody or anti-dengue virus antibody was reduced using DTT, resulting in the breakage of S-S bonds and the generation of S-H groups. The S-H group of the antibody was then further reacted with SPDP-NH-MNBs. The resultant MNBs are termed antibody-integrated MNBs. Abbreviations: DTT, dithiothreitol; MNBs, magnetic nanobeads; SPDP, N -succinimidyl 3-(2-pyridyldithio)propionate.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Schematic representation of Ar/NH 3 plasma-treated MNBs and their binding to anti-virus antibody. Notes: Graphite-encapsulated MNBs were treated with ammonia plasma and modified with amino groups. SPDP was reacted with the amino group of modified MNBs (NH 2 -beads) at pH 7–8. Anti-rotavirus antibody or anti-dengue virus antibody was reduced using DTT, resulting in the breakage of S-S bonds and the generation of S-H groups. The S-H group of the antibody was then further reacted with SPDP-NH-MNBs. The resultant MNBs are termed antibody-integrated MNBs. Abbreviations: DTT, dithiothreitol; MNBs, magnetic nanobeads; SPDP, N -succinimidyl 3-(2-pyridyldithio)propionate.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Binding Assay, Modification

    Schematic representation showing the capture of rotavirus using antibody-integrated MNBs. Notes: Antibody-integrated MNBs (10 µL) were washed twice with PBS. The rotavirus-infected cell lysate (10 µL) was diluted with 500 µL of PBS and used as rotavirus suspension (Step 1). The suspension was then incubated with the MNBs for 5 minutes at room temperature (Step 2). Tubes containing the MNBs were placed in a magnetic field (Step 3). The beads were then subjected to magnetic separation, and the supernatant removed (Step 4). The beads were washed three times with PBS and resuspended in 10 µL PBS for further analysis. Steps 1–4 produced a bead fraction (BD) (10 µL antibody-integrated MNBs following incubation with PBS-diluted rotavirus-infected cell lysate), and a supernatant fraction (SP) (10 µL supernatant; following incubation and washing). Abbreviation: MNBs, magnetic nanobeads.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Schematic representation showing the capture of rotavirus using antibody-integrated MNBs. Notes: Antibody-integrated MNBs (10 µL) were washed twice with PBS. The rotavirus-infected cell lysate (10 µL) was diluted with 500 µL of PBS and used as rotavirus suspension (Step 1). The suspension was then incubated with the MNBs for 5 minutes at room temperature (Step 2). Tubes containing the MNBs were placed in a magnetic field (Step 3). The beads were then subjected to magnetic separation, and the supernatant removed (Step 4). The beads were washed three times with PBS and resuspended in 10 µL PBS for further analysis. Steps 1–4 produced a bead fraction (BD) (10 µL antibody-integrated MNBs following incubation with PBS-diluted rotavirus-infected cell lysate), and a supernatant fraction (SP) (10 µL supernatant; following incubation and washing). Abbreviation: MNBs, magnetic nanobeads.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Infection, Incubation, Produced

    Detection of viral protein 6 (VP6) in rotavirus absorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and incubated with antibody-integrated MNBs prior to magnetic separation. The presence of VP6 was interpreted on the basis of the presence and absence of a test line (Test). A positive control line was also included (Control). Samples were divided into the following categories: diluted rotavirus sample before incubation with the beads (BF), bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). All fractions were solubilized with lysis buffer and subjected to immunochromatography (Immuno Card STAT! ® Rotavirus, Meridian Bioscience Inc., Cincinnati, OH, USA) for the detection of rotavirus VP6. Abbreviation: MNBs, magnetic nanobeads.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Detection of viral protein 6 (VP6) in rotavirus absorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and incubated with antibody-integrated MNBs prior to magnetic separation. The presence of VP6 was interpreted on the basis of the presence and absence of a test line (Test). A positive control line was also included (Control). Samples were divided into the following categories: diluted rotavirus sample before incubation with the beads (BF), bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). All fractions were solubilized with lysis buffer and subjected to immunochromatography (Immuno Card STAT! ® Rotavirus, Meridian Bioscience Inc., Cincinnati, OH, USA) for the detection of rotavirus VP6. Abbreviation: MNBs, magnetic nanobeads.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Infection, Incubation, Positive Control, Lysis

    Quantitative analysis of the viral gene of rotavirus adsorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to RT-reaction. The resultant cDNA was analyzed by real-time PCR using primers for the rotavirus VP7 gene as described in Materials and methods. The value of the TL sample was taken as 100%. Abbreviations: MNBs, magnetic nanobeads; RT, reverse transcription.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Quantitative analysis of the viral gene of rotavirus adsorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to RT-reaction. The resultant cDNA was analyzed by real-time PCR using primers for the rotavirus VP7 gene as described in Materials and methods. The value of the TL sample was taken as 100%. Abbreviations: MNBs, magnetic nanobeads; RT, reverse transcription.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Infection, Incubation, Magnetic Beads, Real-time Polymerase Chain Reaction

    Detection of viral RNA of rotavirus adsorbed onto antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to a RT-reaction. Rotavirus viral protein 7 (VP7) gene (552 bp) in the cDNA was amplified by PCR as described in Materials and methods. PCR products were analyzed by agarose gel electrophoresis (1.2% gel). The identity of the amplified products was confirmed by DNA sequencing. The left-hand lane is size marker (M), which includes DNA of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, and 1,500 bp. The position of the 552 bp band for VP7 is indicated by an arrow. The NC comprised a water sample (no rotavirus) that was subjected to RT-PCR. Abbreviations: MNBs, magnetic nanobeads; NC, negative control; RT, reverse transcription.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Detection of viral RNA of rotavirus adsorbed onto antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to a RT-reaction. Rotavirus viral protein 7 (VP7) gene (552 bp) in the cDNA was amplified by PCR as described in Materials and methods. PCR products were analyzed by agarose gel electrophoresis (1.2% gel). The identity of the amplified products was confirmed by DNA sequencing. The left-hand lane is size marker (M), which includes DNA of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, and 1,500 bp. The position of the 552 bp band for VP7 is indicated by an arrow. The NC comprised a water sample (no rotavirus) that was subjected to RT-PCR. Abbreviations: MNBs, magnetic nanobeads; NC, negative control; RT, reverse transcription.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Infection, Incubation, Magnetic Beads, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Sequencing, Marker, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Infectivity in recovered rotavirus adsorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated MNBs. Fractions are as follows: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). All fractions were subjected to an infection analysis using MA104 cells and IFA, as described in Figure S1. The number of rotavirus-infected cells was calculated as described in Materials and methods. Abbreviations: IFA, indirect fluorescent assay; MNBs, magnetic nanobeads.

    Journal: International Journal of Nanomedicine

    Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

    doi: 10.2147/IJN.S191784

    Figure Lengend Snippet: Infectivity in recovered rotavirus adsorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated MNBs. Fractions are as follows: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). All fractions were subjected to an infection analysis using MA104 cells and IFA, as described in Figure S1. The number of rotavirus-infected cells was calculated as described in Materials and methods. Abbreviations: IFA, indirect fluorescent assay; MNBs, magnetic nanobeads.

    Article Snippet: By using data on the number of rotavirus-infected cells and the number of uninfected cells, we calculated the recovery rate and the concentrating rate as follows: % recovery rate = 100 × ( S / T ) Concentrating rate = S / B where T = quantity of genomic RNA or infectivity of total sample containing the same quantity (10 µL) of rotavirus-infected cell lysate as BD (TL); S = quantity of genomic RNA or infectivity of 10 µL of antibody-integrated MNBs after incubation with diluted rotavirus-infected cell lysate (RV-BD or DV-BD); and B = quantity of genomic RNA or infectivity of 10 µL of rotavirus cell lysate with 500 µL of PBS before incubation with the beads (BF).

    Techniques: Infection, Incubation, Immunofluorescence, Fluorescence

    Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity. In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2 ). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity. In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also Fig 2 ). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Adsorption, Staining, Incubation, Nickel Column, Plasmid Preparation, Expressing, Labeling

    Macrophage/microglial cells infiltrate the Purkinje cell layer after cell death has already begun. The Purkinje cell layer is shown at three points in time. Cells of macrophage/monocyte lineage were not detected within the Purkinje cell layer at 48 hours, or at 72 hours, when cell death was already apparent. Macrophage/mononuclear infiltration did not begin until 96 hours (data not shown) and was more extensive at 120 hours. These data indicate that initiation of Purkinje cell death did not involve antibody-mediated cellular cytotoxicity. Scale bar = 20μ.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Macrophage/microglial cells infiltrate the Purkinje cell layer after cell death has already begun. The Purkinje cell layer is shown at three points in time. Cells of macrophage/monocyte lineage were not detected within the Purkinje cell layer at 48 hours, or at 72 hours, when cell death was already apparent. Macrophage/mononuclear infiltration did not begin until 96 hours (data not shown) and was more extensive at 120 hours. These data indicate that initiation of Purkinje cell death did not involve antibody-mediated cellular cytotoxicity. Scale bar = 20μ.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques:

    Rat cerebellar slice culture incubated with serum from Patient 3, a neurologically normal individual with mixed mesodermal ovarian sarcoma. Panels A and B demonstrate. extensive accumulation of IgG not only within Purkinje cells (arrow) but also in other neurons within and near the Purkinje cell layer (asterisks). Panels C and D : Purkinje and other neurons exhibiting Cy5 labeling for human IgG do not contain SYTOX green, indicating that IgG uptake had occurred in viable cells. Scale bars = 20μ.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Rat cerebellar slice culture incubated with serum from Patient 3, a neurologically normal individual with mixed mesodermal ovarian sarcoma. Panels A and B demonstrate. extensive accumulation of IgG not only within Purkinje cells (arrow) but also in other neurons within and near the Purkinje cell layer (asterisks). Panels C and D : Purkinje and other neurons exhibiting Cy5 labeling for human IgG do not contain SYTOX green, indicating that IgG uptake had occurred in viable cells. Scale bars = 20μ.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Incubation, Labeling

    Comparison of uptake and cytotoxicity of anti-Yo antibody versus anti-Purkinje cell cytoplasmic antibodies obtained from three neurologically normal ovarian carcinoma patients whose sera did not react with Yo antigens. Uptake and accumulation of IgG (red) is seen with anti-Yo IgG and with IgGs from all three neurologically normal patients. Entry of SYTOX green into Purkinje cells containing IgG (yellow), indicative of cell membrane injury and death, is seen only in cultures incubated with anti-Yo IgGs for 96 hours (examples shown by arrows) but not with IgGs from neurologically normal Patients 1, 2, or 3. Uptake of normal IgG is not seen at confocal gain settings used for optimal visualization of anti-Yo antibody accumulation (13). Images shown are representative of three experiments. By the end of incubation (140 hours) staining for SYTOX green, indicative of necrotic cell death, was observed in less than 10% of cells incubated with sera from Patients 1, 2, and 3, an amount of background cell death staining similar to that seen in cultures incubated with control sera (data not shown). Parallel cultures incubated with anti-Yo sera or sera from Patients 1–3 sera remained negative by FLICA staining as compared to controls, indicating that with exposure to these sera did not result in apoptotic cell death (data not shown). Scale bar = 20μ.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Comparison of uptake and cytotoxicity of anti-Yo antibody versus anti-Purkinje cell cytoplasmic antibodies obtained from three neurologically normal ovarian carcinoma patients whose sera did not react with Yo antigens. Uptake and accumulation of IgG (red) is seen with anti-Yo IgG and with IgGs from all three neurologically normal patients. Entry of SYTOX green into Purkinje cells containing IgG (yellow), indicative of cell membrane injury and death, is seen only in cultures incubated with anti-Yo IgGs for 96 hours (examples shown by arrows) but not with IgGs from neurologically normal Patients 1, 2, or 3. Uptake of normal IgG is not seen at confocal gain settings used for optimal visualization of anti-Yo antibody accumulation (13). Images shown are representative of three experiments. By the end of incubation (140 hours) staining for SYTOX green, indicative of necrotic cell death, was observed in less than 10% of cells incubated with sera from Patients 1, 2, and 3, an amount of background cell death staining similar to that seen in cultures incubated with control sera (data not shown). Parallel cultures incubated with anti-Yo sera or sera from Patients 1–3 sera remained negative by FLICA staining as compared to controls, indicating that with exposure to these sera did not result in apoptotic cell death (data not shown). Scale bar = 20μ.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Incubation, Staining

    Comparison of Purkinje cell cytotoxicity produced by anti-Yo antibodies with cytotoxicity produced by commercial and patient antibodies reactive with other Purkinje cell cytoplasmic proteins. Rat cerebellar slice cultures were incubated for 72 hours with either 1) sera from patients with anti-Yo antibodies; 2) commercial antibodies to calbindin, calmodulin, or PCP-2/L7; 3) anti-Purkinje cells antibodies from the three neurologically normal patients studied, whose sera labeled Purkinje cell cytoplasm but did not react with Yo antigens; or 4) normal human IgG. Cultures were quantified for cell death as indicated by uptake of SYTOX dyes. Extensive Purkinje cell death was seen in cultures incubated with all 4 anti-Yo sera (data for 2 anti-Yo sera not shown). In contrast, cell death was not observed in cultures incubated with any of the three commercially obtained antibodies reactive with intracellular anti-Purkinje cell proteins nor with sera from Patients 1–3, despite extensive Purkinje cell uptake. Cultures incubated with normal human IgG exhibited only faint antibody accumulation by Purkinje cells and no detectable Purkinje cell death. Statistical significance between groups was determined by non-parametric Mann-Whitney ANOVA. Death in cultures incubated with anti-Yo antisera was statistically significantly greater than that seen in cultures incubated with commercial antisera, sera from patients 1–3, or normal human IgG.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Comparison of Purkinje cell cytotoxicity produced by anti-Yo antibodies with cytotoxicity produced by commercial and patient antibodies reactive with other Purkinje cell cytoplasmic proteins. Rat cerebellar slice cultures were incubated for 72 hours with either 1) sera from patients with anti-Yo antibodies; 2) commercial antibodies to calbindin, calmodulin, or PCP-2/L7; 3) anti-Purkinje cells antibodies from the three neurologically normal patients studied, whose sera labeled Purkinje cell cytoplasm but did not react with Yo antigens; or 4) normal human IgG. Cultures were quantified for cell death as indicated by uptake of SYTOX dyes. Extensive Purkinje cell death was seen in cultures incubated with all 4 anti-Yo sera (data for 2 anti-Yo sera not shown). In contrast, cell death was not observed in cultures incubated with any of the three commercially obtained antibodies reactive with intracellular anti-Purkinje cell proteins nor with sera from Patients 1–3, despite extensive Purkinje cell uptake. Cultures incubated with normal human IgG exhibited only faint antibody accumulation by Purkinje cells and no detectable Purkinje cell death. Statistical significance between groups was determined by non-parametric Mann-Whitney ANOVA. Death in cultures incubated with anti-Yo antisera was statistically significantly greater than that seen in cultures incubated with commercial antisera, sera from patients 1–3, or normal human IgG.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Produced, Incubation, Labeling, MANN-WHITNEY

    Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2. The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2. The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Incubation, Staining

    Quantitative analysis of Purkinje cell death in rat cerebellar slice cultures incubated with native anti-Yo serum (native serum), sham adsorbed serum, and serum adsorbed with the 62 kDa major Yo antigen. The per cent of Purkinje cells which contained IgG and stained with SYTOX green was counted from eight fields similar to that seen in Fig 1 . Adsorption with the 62 kDa Yo expression protein effectively abolished Purkinje cell death compared to untreated serum or sham-adsorbed serum circulated through a nickel column with bound vector lacking Yo antigen.

    Journal: PLoS ONE

    Article Title: Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    doi: 10.1371/journal.pone.0123446

    Figure Lengend Snippet: Quantitative analysis of Purkinje cell death in rat cerebellar slice cultures incubated with native anti-Yo serum (native serum), sham adsorbed serum, and serum adsorbed with the 62 kDa major Yo antigen. The per cent of Purkinje cells which contained IgG and stained with SYTOX green was counted from eight fields similar to that seen in Fig 1 . Adsorption with the 62 kDa Yo expression protein effectively abolished Purkinje cell death compared to untreated serum or sham-adsorbed serum circulated through a nickel column with bound vector lacking Yo antigen.

    Article Snippet: Sera from patient 3, an individual with a mixed mesodermal sarcoma, produced nuclear and cytoplasmic labeling of Purkinje cells, basket cells, and occasional granule cells; and labeled proteins of 66, 80, and 120 kDa in Western blots of Purkinje cell lysates [ ] (Greenlee et al. unpublished data).

    Techniques: Incubation, Staining, Adsorption, Expressing, Nickel Column, Plasmid Preparation

    Effects of DOC-1R expression on the regulation of G1 phase-related gene expressions. (A) Overexpression of DOC-1R. pFLAG-DOC-1R was transfected into HeLa cells and 48 h after transfection, cells were extracted and subjected to Western blot analysis of CDK2, cyclin D1, cyclin E, p21, p27 and p53 protein, respectively. (B) Knockdown of endogenous DOC-1R. DOC-1R shRNA plasmid was transfected into HEK-293 cells and 48 h post-transfection, cells were extracted and subjected to Western blot analysis of related proteins. A scrambled shRNA plasmid was used as a control.

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: Effects of DOC-1R expression on the regulation of G1 phase-related gene expressions. (A) Overexpression of DOC-1R. pFLAG-DOC-1R was transfected into HeLa cells and 48 h after transfection, cells were extracted and subjected to Western blot analysis of CDK2, cyclin D1, cyclin E, p21, p27 and p53 protein, respectively. (B) Knockdown of endogenous DOC-1R. DOC-1R shRNA plasmid was transfected into HEK-293 cells and 48 h post-transfection, cells were extracted and subjected to Western blot analysis of related proteins. A scrambled shRNA plasmid was used as a control.

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Expressing, Over Expression, Transfection, Western Blot, shRNA, Plasmid Preparation

    Interaction between DOC-1R and CDK2 proteins. (A) GST pull-down assay. GST or GST-DOC-1R proteins were expressed in Escherichia coli BL21, extracted, incubated with total lysates of HeLa cells and MagneGST particles, and then subjected to Western blot analysis of CDK2, cyclin A and cyclin E, respectively. (B) Immunoprecipitation-Western blot assay. Recombinant retrovirus carrying DOC-1R cDNA was produced and used to infect HeLa cells. Immunoprecipitation was performed as described above following with Western blot analysis. (C) Fluorescent assay. pEGFP-DOC-1R or control was transfected into CHO cells with pDsRED-CDK2. Forty-eight hours later, cell images were viewed and analyzed with a fluorescent microscope.

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: Interaction between DOC-1R and CDK2 proteins. (A) GST pull-down assay. GST or GST-DOC-1R proteins were expressed in Escherichia coli BL21, extracted, incubated with total lysates of HeLa cells and MagneGST particles, and then subjected to Western blot analysis of CDK2, cyclin A and cyclin E, respectively. (B) Immunoprecipitation-Western blot assay. Recombinant retrovirus carrying DOC-1R cDNA was produced and used to infect HeLa cells. Immunoprecipitation was performed as described above following with Western blot analysis. (C) Fluorescent assay. pEGFP-DOC-1R or control was transfected into CHO cells with pDsRED-CDK2. Forty-eight hours later, cell images were viewed and analyzed with a fluorescent microscope.

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Pull Down Assay, Incubation, Western Blot, Immunoprecipitation, Recombinant, Produced, Fluorescence, Transfection, Microscopy

    Inhibition of CDK2 expression at the transcriptional level by overexpression of DOC-1R. (A) DOC-1R overexpression down-regulated CDK2 expression via an ubiquitin-independent manner. HeLa cells were transfected with DOC-1R or control plasmid in presence or absence of the proteosome inhibitor MG-132 (0.01 mM) for 8 h and then subjected to Western blot analysis of CDK2 or FLAG-DOC-1R. (B, C) DOC-1R overexpression reduced CDK2 mRNA levels. Cells cultured in the same conditions were subjected to RNA isolation and then RT-PCR (B) and qRT-PCR (C) to detect CDK2 expression.

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: Inhibition of CDK2 expression at the transcriptional level by overexpression of DOC-1R. (A) DOC-1R overexpression down-regulated CDK2 expression via an ubiquitin-independent manner. HeLa cells were transfected with DOC-1R or control plasmid in presence or absence of the proteosome inhibitor MG-132 (0.01 mM) for 8 h and then subjected to Western blot analysis of CDK2 or FLAG-DOC-1R. (B, C) DOC-1R overexpression reduced CDK2 mRNA levels. Cells cultured in the same conditions were subjected to RNA isolation and then RT-PCR (B) and qRT-PCR (C) to detect CDK2 expression.

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Inhibition, Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    DOC-1R expression inhibited G1/S transition and suppressed CDK2 kinase activity. (A) Cell cycle analysis. HeLa cells were transiently transfected with pFLAG-DOC-1R and then subjected to flow cytometry assay at 48 h post-transfection. * P

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: DOC-1R expression inhibited G1/S transition and suppressed CDK2 kinase activity. (A) Cell cycle analysis. HeLa cells were transiently transfected with pFLAG-DOC-1R and then subjected to flow cytometry assay at 48 h post-transfection. * P

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Expressing, Activity Assay, Cell Cycle Assay, Transfection, Flow Cytometry, Cytometry

    DOC-1R-mediated inhibition of CDK2 activation through repressing cyclin E/A-CDK2 interactions. (A) Western blot. HeLa cells were transfected with pFLAG-DOC-1R and then subjected to protein extraction and western blot analysis for total CDK2, pCDK2 (Thr160) and pCDK2 (Thr14/Tyr15) proteins, respectively. (B) Immunoprecipitation-Western blot assay. The cells with the same conditions as A were subjected to immunoprecipitation with an anti-CDK2 antibody, as well as Western blot analysis for cyclin E and A protein, respectively.

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: DOC-1R-mediated inhibition of CDK2 activation through repressing cyclin E/A-CDK2 interactions. (A) Western blot. HeLa cells were transfected with pFLAG-DOC-1R and then subjected to protein extraction and western blot analysis for total CDK2, pCDK2 (Thr160) and pCDK2 (Thr14/Tyr15) proteins, respectively. (B) Immunoprecipitation-Western blot assay. The cells with the same conditions as A were subjected to immunoprecipitation with an anti-CDK2 antibody, as well as Western blot analysis for cyclin E and A protein, respectively.

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Inhibition, Activation Assay, Western Blot, Transfection, Protein Extraction, Immunoprecipitation

    Identification of the amino acid region of DOC-1R protein that interacted with CDK2 protein. (A) Schematic representation of the truncated DOC-1R proteins. (B) GST pull-down assay. Truncated DOC-1R proteins were expressed in Escherichia coli BL-21 cells, pulled down with total lysates of HeLa cells and MagneGST particles, and then immunoblotted with anti-CDK2 antibody. (C) Schematic representation of the truncated CDK2 protein. (D) GST pull-down assay. Truncated FLAG-CDK2 proteins were subjected to pull-down assays with recombinant GST-tagged DOC-1R protein and then immunoblotted with anti-FLAG antibody.

    Journal: International Journal of Biological Sciences

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

    doi: 10.7150/ijbs.5763

    Figure Lengend Snippet: Identification of the amino acid region of DOC-1R protein that interacted with CDK2 protein. (A) Schematic representation of the truncated DOC-1R proteins. (B) GST pull-down assay. Truncated DOC-1R proteins were expressed in Escherichia coli BL-21 cells, pulled down with total lysates of HeLa cells and MagneGST particles, and then immunoblotted with anti-CDK2 antibody. (C) Schematic representation of the truncated CDK2 protein. (D) GST pull-down assay. Truncated FLAG-CDK2 proteins were subjected to pull-down assays with recombinant GST-tagged DOC-1R protein and then immunoblotted with anti-FLAG antibody.

    Article Snippet: For the in vitro binding assay, GST, GST-DOC-1R and truncated proteins were gently rotated with HeLa lysates and MagneGST particles (Promega) overnight based on the manufacturer's protocol.

    Techniques: Pull Down Assay, Recombinant

    IVIG causes aggregation of both encapsulated and unencapsulated S . pneumoniae . ( A ) Light microscopy of bacteria (at 100 X with rapid Romanovsky stain) after 8 hr culture of wild-type (TIGR4) and unencapsulated (TIGR4Δcps) bacteria with or without 10% IVIG. (B) Effect of relative concentration of IVIG on size of bacterial aggregate particles after 30 minute incubation of wild-type (open bars) or unencapsulated (filled bars) TIGR4 measured as forward scatter in flow cytometry. P values were calculated using one-way ANOVA. (C) to (F) Changes in optical density at 580nm (OD 580 ) during culture of wild-type (triangles) and unencapsulated (circles) S . pneumoniae (C) TIGR4, (D) ST2 (D39), (E) ST3 (0100993), (F) or ST23F strain in THY broth supplemented with either 10% IVIG (filled symbols) or PBS (empty symbols). (G) Effect of vigorous pipetting (filled symbols) or no pipetting (empty symbols) immediately prior to measurement of OD580 during culture of wild-type (triangles) and unencapsulated (circles) TIGR4 in 10% IVIG. (H) Effect of addition of either 1% IVIG (filled circles) or 10% (upside triangles), 5% (diamonds) or 2% (crosses) papain-treated IVIG, or PBS (empty circles) on OD 580 during culture of unencapsulated TIGR4. For panels B to H, data are presented as means and error bars represent SDs of three to four technical replicates and are representative of experiments repeated at least twice. *, P

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: IVIG causes aggregation of both encapsulated and unencapsulated S . pneumoniae . ( A ) Light microscopy of bacteria (at 100 X with rapid Romanovsky stain) after 8 hr culture of wild-type (TIGR4) and unencapsulated (TIGR4Δcps) bacteria with or without 10% IVIG. (B) Effect of relative concentration of IVIG on size of bacterial aggregate particles after 30 minute incubation of wild-type (open bars) or unencapsulated (filled bars) TIGR4 measured as forward scatter in flow cytometry. P values were calculated using one-way ANOVA. (C) to (F) Changes in optical density at 580nm (OD 580 ) during culture of wild-type (triangles) and unencapsulated (circles) S . pneumoniae (C) TIGR4, (D) ST2 (D39), (E) ST3 (0100993), (F) or ST23F strain in THY broth supplemented with either 10% IVIG (filled symbols) or PBS (empty symbols). (G) Effect of vigorous pipetting (filled symbols) or no pipetting (empty symbols) immediately prior to measurement of OD580 during culture of wild-type (triangles) and unencapsulated (circles) TIGR4 in 10% IVIG. (H) Effect of addition of either 1% IVIG (filled circles) or 10% (upside triangles), 5% (diamonds) or 2% (crosses) papain-treated IVIG, or PBS (empty circles) on OD 580 during culture of unencapsulated TIGR4. For panels B to H, data are presented as means and error bars represent SDs of three to four technical replicates and are representative of experiments repeated at least twice. *, P

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Light Microscopy, Staining, Concentration Assay, Incubation, Flow Cytometry, Cytometry

    IgG binding to live S . pneumoniae after incubation with IVIG. ( A ) Effect of presence (wild-type strain, +cps) or absence (unencapsulated strain, -cps) of the capsule on IgG binding to live S . pneumoniae TIGR4 incubated in either 1% IVIG, 10% IVIG or PBS (control). IgG binding was measured as MFI using anti-IgG-PE by flow cytometry. ( B ) Effect of presence or absence of capsule on IgG binding to 3 other serotype strains (ST2, strain D39; ST23F; ST3, strain 0100993) incubated in 10% IVIG. ( C ) Effect of pre-treatment of bacteria with Pronase or PBS prior to incubation in 10% IVIG on IgG binding to TIGR4, and an example of the histogram for IgG binding MFI (solid line, PBS then IVIG; dotted line PBS then Pronase then IVIG; shaded area, no IVIG). (D) Effect of loss of expression of lipoproteins ( Δlgt ) or both the choline binding proteins PspA and PspC ( ΔpspA/pspC ) on IgG binding to the D39 strain after incubation in 10% IVIG, with an example of the histogram for the MFI of IgG binding (solid line, Δlgt ; dotted line, ΔpspA/pspC ; shaded area, D39). (E) Effect of depletion of specific surface protein antibodies from 10% IVIG using absorption with unencapsulated TIGR4 prior to incubating wild type TIGR4 bacteria in IVIG and measuring IgG binding using flow cytometry. (F) and (G) Effect of expressing potential S . pneumoniae antigens in S . mitis on IgG binding to live bacteria after incubation in 10% IVIG. ( F ) IgG binding to wild-type S . mitis (WT), S . mitis manipulated to lacking its own capsule (Δcps) or expressing S . pneumoniae serotype 4 capsule (T4cps). ( G ) IgG binding to wild-type S . mitis (WT) or S . mitis expressing PspC. For all panels, data are presented as means and SDs of three to four technical replicates and are representative of experiments repeated at least twice. P values were calculated using unpaired 2-tailed Student t-tests.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: IgG binding to live S . pneumoniae after incubation with IVIG. ( A ) Effect of presence (wild-type strain, +cps) or absence (unencapsulated strain, -cps) of the capsule on IgG binding to live S . pneumoniae TIGR4 incubated in either 1% IVIG, 10% IVIG or PBS (control). IgG binding was measured as MFI using anti-IgG-PE by flow cytometry. ( B ) Effect of presence or absence of capsule on IgG binding to 3 other serotype strains (ST2, strain D39; ST23F; ST3, strain 0100993) incubated in 10% IVIG. ( C ) Effect of pre-treatment of bacteria with Pronase or PBS prior to incubation in 10% IVIG on IgG binding to TIGR4, and an example of the histogram for IgG binding MFI (solid line, PBS then IVIG; dotted line PBS then Pronase then IVIG; shaded area, no IVIG). (D) Effect of loss of expression of lipoproteins ( Δlgt ) or both the choline binding proteins PspA and PspC ( ΔpspA/pspC ) on IgG binding to the D39 strain after incubation in 10% IVIG, with an example of the histogram for the MFI of IgG binding (solid line, Δlgt ; dotted line, ΔpspA/pspC ; shaded area, D39). (E) Effect of depletion of specific surface protein antibodies from 10% IVIG using absorption with unencapsulated TIGR4 prior to incubating wild type TIGR4 bacteria in IVIG and measuring IgG binding using flow cytometry. (F) and (G) Effect of expressing potential S . pneumoniae antigens in S . mitis on IgG binding to live bacteria after incubation in 10% IVIG. ( F ) IgG binding to wild-type S . mitis (WT), S . mitis manipulated to lacking its own capsule (Δcps) or expressing S . pneumoniae serotype 4 capsule (T4cps). ( G ) IgG binding to wild-type S . mitis (WT) or S . mitis expressing PspC. For all panels, data are presented as means and SDs of three to four technical replicates and are representative of experiments repeated at least twice. P values were calculated using unpaired 2-tailed Student t-tests.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Binding Assay, Incubation, Flow Cytometry, Cytometry, Expressing

    eIVIG immune recognition of S . pneumoniae . (A-D) Whole cell ELISAs of IgG binding to solid-phase S . pneumoniae of increasing concentrations of eIVIG made using TIGR4 (A and B) or D39 (C and D) or IVIG. Binding was assessed to the solid phase strain that was homologous ( A and D ) or heterologous ( B and C ) to the enrichment strain. ( E-F ) Bacterial surface IgG binding measured by flow cytometry to S . pneumoniae TIGR4 (E) or D39 (F) incubated in either PBS (shaded area) or TIGR4 eIVIG at 30 μg/ml (solid line). P values were calculated using linear regression for panels A, B, C, and D, and unpaired 2-tailed Student t-tests for panels E and F.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: eIVIG immune recognition of S . pneumoniae . (A-D) Whole cell ELISAs of IgG binding to solid-phase S . pneumoniae of increasing concentrations of eIVIG made using TIGR4 (A and B) or D39 (C and D) or IVIG. Binding was assessed to the solid phase strain that was homologous ( A and D ) or heterologous ( B and C ) to the enrichment strain. ( E-F ) Bacterial surface IgG binding measured by flow cytometry to S . pneumoniae TIGR4 (E) or D39 (F) incubated in either PBS (shaded area) or TIGR4 eIVIG at 30 μg/ml (solid line). P values were calculated using linear regression for panels A, B, C, and D, and unpaired 2-tailed Student t-tests for panels E and F.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Incubation

    Effects of unencapsulated and encapsulated S . pneumoniae on IVIG whole cell ELISA titres for four strains. Competitive inhibition of IVIG (1/10000) whole cell ELISAs of IgG binding to the homologous solid-phase S . pneumoniae strain using increasing concentrations of encapsulated or unencapsulated bacterial lysates for wild-type (A) TIGR4, (B) D39, (C) serotype 3, ( D) serotype 23F strains, and for unencapsulated mutant derivative of (E) serotype 3 and (F) D39 strains. Data presented as means and SDs of three technical replicates.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Effects of unencapsulated and encapsulated S . pneumoniae on IVIG whole cell ELISA titres for four strains. Competitive inhibition of IVIG (1/10000) whole cell ELISAs of IgG binding to the homologous solid-phase S . pneumoniae strain using increasing concentrations of encapsulated or unencapsulated bacterial lysates for wild-type (A) TIGR4, (B) D39, (C) serotype 3, ( D) serotype 23F strains, and for unencapsulated mutant derivative of (E) serotype 3 and (F) D39 strains. Data presented as means and SDs of three technical replicates.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Mutagenesis

    Variation of IgG antigen targets between individuals and with age. (A-C) Results for six Malawian subjects (labelled A to G). (A) whole cell ELISAs to 4 S . pneumoniae strains (serotype 4, 14, 9C and 1). (B) Immunoblots of IgG binding to the wild-type S . pneumoniae strain D39 (10 μg / lane, red boxes highlight bands that visibly vary in intensity between subjects). (C) MFI of IgG binding to selected protein antigens measured using a Luminex assay. Data points represent mean (SEMs) of two technical duplicates for IgG binding. For immunoblots and the Luminex assay, sera were diluted to 1/1000. (D) Levels of serum IgG binding to specific protein antigens aged (mean age 67.2 years, black symbols) and young subjects (mean age 31.2 years, empty symbols) measured by multiplex MSD (only results for antigens with stronger responses are shown as means and SEM). (E-F) Levels of serum IgG binding for each individual aged (black symbols) and young (empty symbols) adult subjects to the protein antigens PspC (E) and PcpA (F) measured by MSD. P values were calculated using unpaired T tests, with bars representing means for the group.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Variation of IgG antigen targets between individuals and with age. (A-C) Results for six Malawian subjects (labelled A to G). (A) whole cell ELISAs to 4 S . pneumoniae strains (serotype 4, 14, 9C and 1). (B) Immunoblots of IgG binding to the wild-type S . pneumoniae strain D39 (10 μg / lane, red boxes highlight bands that visibly vary in intensity between subjects). (C) MFI of IgG binding to selected protein antigens measured using a Luminex assay. Data points represent mean (SEMs) of two technical duplicates for IgG binding. For immunoblots and the Luminex assay, sera were diluted to 1/1000. (D) Levels of serum IgG binding to specific protein antigens aged (mean age 67.2 years, black symbols) and young subjects (mean age 31.2 years, empty symbols) measured by multiplex MSD (only results for antigens with stronger responses are shown as means and SEM). (E-F) Levels of serum IgG binding for each individual aged (black symbols) and young (empty symbols) adult subjects to the protein antigens PspC (E) and PcpA (F) measured by MSD. P values were calculated using unpaired T tests, with bars representing means for the group.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Western Blot, Binding Assay, Luminex, Multiplex Assay

    IVIG improves macrophage and neutrophil opsonophagocytosis of both encapsulated an unencapsulated S . pneumoniae TIGR4. ( A ) MFI and example of a histogram of the fluorescence of RAW macrophages incubated with fluorescent (FAM-SE labelled) encapsulated (clear columns) and unencapsulated (black columns) S . pneumoniae TIGR4 strains with and without addition of 10% IVIG. P values were calculated using unpaired 2-tailed Student t-test. (B ) MFI of fluorescence intensity of human neutrophils following incubation with FAM-SE labelled S . pneumoniae TIGR4 encapsulated (clear columns) and unencapsulated (filled columns) strains pre-opsonised with increasing concentrations of IVIG. P values were calculated using one-way ANOVAs. ( C ) Relative survival of S . pneumoniae TIGR4 encapsulated (+cps) and unencapsulated (-cps) strains following incubation with human neutrophils in the presence of 10% IVIG. Percentage survival calculated relative to survival of identical strain incubated without IVIG, with the surviving CFU / ml shown for control versus 10% IVIG above each column. For all panels, data are presented as means and SDs of three to four technical replicates and are representative of experiments repeated at least twice. P values were calculated using unpaired 2-tailed Student t-test ( A and C ) or one way ANOVAs ( B ).

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: IVIG improves macrophage and neutrophil opsonophagocytosis of both encapsulated an unencapsulated S . pneumoniae TIGR4. ( A ) MFI and example of a histogram of the fluorescence of RAW macrophages incubated with fluorescent (FAM-SE labelled) encapsulated (clear columns) and unencapsulated (black columns) S . pneumoniae TIGR4 strains with and without addition of 10% IVIG. P values were calculated using unpaired 2-tailed Student t-test. (B ) MFI of fluorescence intensity of human neutrophils following incubation with FAM-SE labelled S . pneumoniae TIGR4 encapsulated (clear columns) and unencapsulated (filled columns) strains pre-opsonised with increasing concentrations of IVIG. P values were calculated using one-way ANOVAs. ( C ) Relative survival of S . pneumoniae TIGR4 encapsulated (+cps) and unencapsulated (-cps) strains following incubation with human neutrophils in the presence of 10% IVIG. Percentage survival calculated relative to survival of identical strain incubated without IVIG, with the surviving CFU / ml shown for control versus 10% IVIG above each column. For all panels, data are presented as means and SDs of three to four technical replicates and are representative of experiments repeated at least twice. P values were calculated using unpaired 2-tailed Student t-test ( A and C ) or one way ANOVAs ( B ).

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Fluorescence, Incubation

    Antigen targets for IgG in IVIG. ( A ) Immunoblots of IVIG (1/1000) binding to whole cell lysates (10 μg / lane) of S . pneumoniae strains D39, TIGR4, 0100993, ST6B, ST14 and ST23F. ( B ) and ( C ) Competitive inhibition in IVIG (1/10000) whole cell ELISAs of IgG binding to solid-phase S . pneumoniae TIGR4 using increasing concentrations of (B) soluble lysates of TIGR4 with or without pre-treatment with trypsin, or (C) soluble purified cell wall polysaccharide (CWPS), soluble serotype 4 capsule (CPS), or soluble S . pneumoniae TIGR4 lysates. Data presented as means and SDs of four technical replicates and are representative of experiments repeated twice. P values calculated using 2-way ANOVAs and Bonferroni post-test comparisons; error bars represent SD.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Antigen targets for IgG in IVIG. ( A ) Immunoblots of IVIG (1/1000) binding to whole cell lysates (10 μg / lane) of S . pneumoniae strains D39, TIGR4, 0100993, ST6B, ST14 and ST23F. ( B ) and ( C ) Competitive inhibition in IVIG (1/10000) whole cell ELISAs of IgG binding to solid-phase S . pneumoniae TIGR4 using increasing concentrations of (B) soluble lysates of TIGR4 with or without pre-treatment with trypsin, or (C) soluble purified cell wall polysaccharide (CWPS), soluble serotype 4 capsule (CPS), or soluble S . pneumoniae TIGR4 lysates. Data presented as means and SDs of four technical replicates and are representative of experiments repeated twice. P values calculated using 2-way ANOVAs and Bonferroni post-test comparisons; error bars represent SD.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Western Blot, Binding Assay, Inhibition, Purification

    Absorption of anti-capsular antibody does not prevent IVIG protection against murine models of IPD. (A) Mean (SD) anti-PsaA titres measured by ELISA in IVIG following depletion of type 4 specific IgG by pre-absorption with type 4 capsule expressing S . mitis . ( B ) Mean (SD) anti-capsular serotype 4 titre IgG titre measured by ELISA in type 4 specific IgG-depleted IVIG. ( B ) Mean (SD) MFI of anti-human IgG binding to wild-type (clear columns) and unencapsulated (black columns) S . pneumoniae TIGR4 following incubation in 1% or 10% in type 4 specific IgG-depleted IVIG. ( C ) Bacterial surface IgG binding measured by flow cytometry to S . pneumoniae TIGR4 incubated in PBS (grey column), 1% or 10% mock absorbed IVIG with (filled columns) or type 4 specific IgG-depleted IVIG (open columns). For panels (A) to (C) data are presented as means and SDs of three to four technical replicates and representative of experiments repeated at least twice. ( D ) Log 10 CFU recovered from BALF, lungs, and blood 24 h after i.n. challenge with 1x10 7 CFU S . pneumoniae TIGR4 for mice pre-treated with 12.8 mg of type 4 specific IgG-depleted IVIG or PBS. ( E ) Log 10 CFU recovered from blood 4 h after i.v. challenge with 5x10 5 CFU S . pneumoniae TIGR4 for mice pre-treated with PBS (open circles), or 12.8 mg of mock absorbed IVIG (black circles) or type 4 specific IgG-depleted IVIG (grey circles). Data for panels (D) and (E) were obtained using IVIG depleted of capsular antibody on separate occasions; symbols represent data from individual mice, bars represent group means, and P values were calculated using unpaired 2-tail Student t-test (D) or one way ANOVA (E). Dashed lines represent the limit of detection.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Absorption of anti-capsular antibody does not prevent IVIG protection against murine models of IPD. (A) Mean (SD) anti-PsaA titres measured by ELISA in IVIG following depletion of type 4 specific IgG by pre-absorption with type 4 capsule expressing S . mitis . ( B ) Mean (SD) anti-capsular serotype 4 titre IgG titre measured by ELISA in type 4 specific IgG-depleted IVIG. ( B ) Mean (SD) MFI of anti-human IgG binding to wild-type (clear columns) and unencapsulated (black columns) S . pneumoniae TIGR4 following incubation in 1% or 10% in type 4 specific IgG-depleted IVIG. ( C ) Bacterial surface IgG binding measured by flow cytometry to S . pneumoniae TIGR4 incubated in PBS (grey column), 1% or 10% mock absorbed IVIG with (filled columns) or type 4 specific IgG-depleted IVIG (open columns). For panels (A) to (C) data are presented as means and SDs of three to four technical replicates and representative of experiments repeated at least twice. ( D ) Log 10 CFU recovered from BALF, lungs, and blood 24 h after i.n. challenge with 1x10 7 CFU S . pneumoniae TIGR4 for mice pre-treated with 12.8 mg of type 4 specific IgG-depleted IVIG or PBS. ( E ) Log 10 CFU recovered from blood 4 h after i.v. challenge with 5x10 5 CFU S . pneumoniae TIGR4 for mice pre-treated with PBS (open circles), or 12.8 mg of mock absorbed IVIG (black circles) or type 4 specific IgG-depleted IVIG (grey circles). Data for panels (D) and (E) were obtained using IVIG depleted of capsular antibody on separate occasions; symbols represent data from individual mice, bars represent group means, and P values were calculated using unpaired 2-tail Student t-test (D) or one way ANOVA (E). Dashed lines represent the limit of detection.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Binding Assay, Incubation, Flow Cytometry, Cytometry, Mouse Assay

    Identification of protein antigens recognised by different sources of pooled IgG. ( A ) Immunoblots of IgG (1/1000) binding to the wild-type S . pneumoniae strain D39 and isogenic mutant strains (10 μg) lacking specific surface proteins using IVIG. Boxes highlight missing bands corresponding to the molecular weight for the protein(s) absent in the mutant strains. ( B ) Immunoblots of IgG binding to selected recombinant S . pneumoniae proteins (0.5 μg / lane) probed with IVIG (1/500). ( C ) Immunoblots of IgG binding to wild-type S . pneumoniae D39 strain (10 μg / lane) using pooled sera from different geographical regions, commercial IVIG (1/3300) from Europe (Intratect) or USA (Vigam) and from Malawi. ( D ) Linear regression of the rank order of strength of IgG binding to different protein antigens between serum pooled from donors from Malawi and IVIG preparations obtained from the USA (Vigam) or Europe (Intratect). Data points represent the rank order of each protein antigen for the mean MFI of two technical duplicates for IgG binding measured using the Luminex assay. P and R 2 values were obtained using F tests.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Identification of protein antigens recognised by different sources of pooled IgG. ( A ) Immunoblots of IgG (1/1000) binding to the wild-type S . pneumoniae strain D39 and isogenic mutant strains (10 μg) lacking specific surface proteins using IVIG. Boxes highlight missing bands corresponding to the molecular weight for the protein(s) absent in the mutant strains. ( B ) Immunoblots of IgG binding to selected recombinant S . pneumoniae proteins (0.5 μg / lane) probed with IVIG (1/500). ( C ) Immunoblots of IgG binding to wild-type S . pneumoniae D39 strain (10 μg / lane) using pooled sera from different geographical regions, commercial IVIG (1/3300) from Europe (Intratect) or USA (Vigam) and from Malawi. ( D ) Linear regression of the rank order of strength of IgG binding to different protein antigens between serum pooled from donors from Malawi and IVIG preparations obtained from the USA (Vigam) or Europe (Intratect). Data points represent the rank order of each protein antigen for the mean MFI of two technical duplicates for IgG binding measured using the Luminex assay. P and R 2 values were obtained using F tests.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Western Blot, Binding Assay, Mutagenesis, Molecular Weight, Recombinant, Luminex

    Passive vaccination with IVIG protects against IPD in murine models of S . pneumoniae infection. Mice were passively vaccinated by i.p. administration of 12.8 mg of IVIG (Intratect) or PBS 3 hours before challenge with S . pneumoniae . ( A ) and (B) Concentration of human IgG measured by ELISA after (A) in mouse sera 3 h post-IVIG (n = 4), and ( B ) in mouse BALF recovered from mice 3 h post-IVIG and immediately before and at 2.5 and 24 h after i.n. challenge with 10 7 CFU of TIGR4 strain S . pneumoniae (n = 4 to 6). (C) Correlation between concentration of murine albumin (mg/ml) and human IgG (μg/ml) in BALF 24 hr following invasive i.n. challenge in IVIG-treated mice (n = 6); P and r 2 values were calculated using the F test. (D) Bacterial CFU (log 10 ) recovered from BALF 2.5 hr following IN challenge with 5x10 5 CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6 or 7). (E) Bacterial CFU (log 10 ) recovered from BALF, lung tissue or blood 24 hr following IN challenge with 10 7 CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6). (F) Bacterial CFU (log 10 ) recovered from blood 4 hr following i.v. challenge with 5x10 5 CFU of TIGR4 of IVIG-treated or PBS-treated mice (n = 5). (G) Effect of administration of i.v. 100 μl liposomal clodronate (5mg/ml) to mice on the numbers of F4/80+ve splenocytes measured by flow cytometry (n = 6), with data presented as means, error bars represent SDs, and P values were calculated using unpaired 2-tail Student t-test. (H) Effect of clodronate or PBS administration on bacterial CFU (log 10 ) recovered from the blood of IVIG-treated mice 4 hr following i.v. challenge with 5x10 5 CFU of TIGR4 (n = 5). ( I) Bacterial CFU (log 10 ) recovered from the lungs of neutrophil depleted mice (by prior treatment with the antiLy6 antibody 1A8) given either IVIG or PBS 24 hr and then inoculated i.n. with 5x10 6 CFU of TIGR4 (n = 11 or 12). For (A, B, D, E, F, H, I) , symbols represent data from individual mice, bars represent group means, and P values were calculated using unpaired 2-tail Student t-test. Dashed lines represent the limit of detection.

    Journal: PLoS Pathogens

    Article Title: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    doi: 10.1371/journal.ppat.1006137

    Figure Lengend Snippet: Passive vaccination with IVIG protects against IPD in murine models of S . pneumoniae infection. Mice were passively vaccinated by i.p. administration of 12.8 mg of IVIG (Intratect) or PBS 3 hours before challenge with S . pneumoniae . ( A ) and (B) Concentration of human IgG measured by ELISA after (A) in mouse sera 3 h post-IVIG (n = 4), and ( B ) in mouse BALF recovered from mice 3 h post-IVIG and immediately before and at 2.5 and 24 h after i.n. challenge with 10 7 CFU of TIGR4 strain S . pneumoniae (n = 4 to 6). (C) Correlation between concentration of murine albumin (mg/ml) and human IgG (μg/ml) in BALF 24 hr following invasive i.n. challenge in IVIG-treated mice (n = 6); P and r 2 values were calculated using the F test. (D) Bacterial CFU (log 10 ) recovered from BALF 2.5 hr following IN challenge with 5x10 5 CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6 or 7). (E) Bacterial CFU (log 10 ) recovered from BALF, lung tissue or blood 24 hr following IN challenge with 10 7 CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6). (F) Bacterial CFU (log 10 ) recovered from blood 4 hr following i.v. challenge with 5x10 5 CFU of TIGR4 of IVIG-treated or PBS-treated mice (n = 5). (G) Effect of administration of i.v. 100 μl liposomal clodronate (5mg/ml) to mice on the numbers of F4/80+ve splenocytes measured by flow cytometry (n = 6), with data presented as means, error bars represent SDs, and P values were calculated using unpaired 2-tail Student t-test. (H) Effect of clodronate or PBS administration on bacterial CFU (log 10 ) recovered from the blood of IVIG-treated mice 4 hr following i.v. challenge with 5x10 5 CFU of TIGR4 (n = 5). ( I) Bacterial CFU (log 10 ) recovered from the lungs of neutrophil depleted mice (by prior treatment with the antiLy6 antibody 1A8) given either IVIG or PBS 24 hr and then inoculated i.n. with 5x10 6 CFU of TIGR4 (n = 11 or 12). For (A, B, D, E, F, H, I) , symbols represent data from individual mice, bars represent group means, and P values were calculated using unpaired 2-tail Student t-test. Dashed lines represent the limit of detection.

    Article Snippet: To assess whether there is significant variation between individuals in which S . pneumoniae antigens are recognised by naturally acquired IgG, whole cell ELISAs to four S . pneumoniae serotypes, immunoblots against S . pneumoniae lysates, the Luminex assay of protein antigen responses, and capsular serotype antibody ELISAs were repeated using sera from six young adult HIV negative Malawian individuals (mean age 29 years, range 21 to 36, 3 male, 3 female).

    Techniques: Infection, Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    Effects of CSZ on phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK and cyclic adenosine monophosphate response element-binding protein (CREB) in Aβ-stimulated SH-SY5Y cells. Phosphorylation of ERK1/2, p38 MAPK and CREB were examined using ELISA kit described in “Materials and Methods” section. (A) ERK1/2 phosphorylation. (B) p38 MAPK phosphorylation. SH-SY5Y cells were incubated with or without pretreatment with CSZ for 1 h followed by treatment with Aβ for 30 min. The effects of respective inhibitor were also examined. (C) CREB phosphorylation. Values are expressed as mean + SEM # Compared vs. respective control cells ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effects of CSZ on phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK and cyclic adenosine monophosphate response element-binding protein (CREB) in Aβ-stimulated SH-SY5Y cells. Phosphorylation of ERK1/2, p38 MAPK and CREB were examined using ELISA kit described in “Materials and Methods” section. (A) ERK1/2 phosphorylation. (B) p38 MAPK phosphorylation. SH-SY5Y cells were incubated with or without pretreatment with CSZ for 1 h followed by treatment with Aβ for 30 min. The effects of respective inhibitor were also examined. (C) CREB phosphorylation. Values are expressed as mean + SEM # Compared vs. respective control cells ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    Effects of CSZ on Cu/Zn-Superoxide Dismutase (SOD) content, SOD activity in Aβ-stimulated SH-SY5Y cells. (A) Cu/Zn-SOD isozyme contents, (B) SOD activities in the cell lysate were determined. The effect of a MEK1/2 or p38 MAPK inhibitor was also examined. Values are expressed as mean + SEM. # Statistical significance compared vs. control cells ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effects of CSZ on Cu/Zn-Superoxide Dismutase (SOD) content, SOD activity in Aβ-stimulated SH-SY5Y cells. (A) Cu/Zn-SOD isozyme contents, (B) SOD activities in the cell lysate were determined. The effect of a MEK1/2 or p38 MAPK inhibitor was also examined. Values are expressed as mean + SEM. # Statistical significance compared vs. control cells ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Activity Assay

    Effects of CSZ on caspase-3 and -9 activitives in Aβ-stimulated SH-SY5Y cells. Caspase-3 and -9 activities were measured using each peptide substrate (DEVD-AFC and LEHD-AFC). (A) Caspase-3 activity in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 20 h. (B) Caspase-3 activity was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Caspase-3 activity of the control cells was 60.54 ± 4.35 nmol/mg protein/h. (C) Caspase-9 activity in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 20 h. (D) Caspase-9 activity was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Caspase-9 activity of the control cells was 25.72 ± 3.2 nmol/mg protein/h. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effects of CSZ on caspase-3 and -9 activitives in Aβ-stimulated SH-SY5Y cells. Caspase-3 and -9 activities were measured using each peptide substrate (DEVD-AFC and LEHD-AFC). (A) Caspase-3 activity in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 20 h. (B) Caspase-3 activity was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Caspase-3 activity of the control cells was 60.54 ± 4.35 nmol/mg protein/h. (C) Caspase-9 activity in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 20 h. (D) Caspase-9 activity was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Caspase-9 activity of the control cells was 25.72 ± 3.2 nmol/mg protein/h. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Activity Assay, Incubation

    Effect of CSZ on reactive oxygen species (ROS) generation in Aβ-stimulated SH-SY5Y cells. ROS generation in SH-SY5Y cells was evaluated using CM-H2DCFDA. (A) The generation of ROS in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 1 h. (B) The generation of ROS was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or 10 μM inhibitor of MEK1/2 or p38 MAPK followed by 1 h incubation with Aβ. ROS generation of the control cells was 1455290 ± 70201 Fluorescence intensity. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effect of CSZ on reactive oxygen species (ROS) generation in Aβ-stimulated SH-SY5Y cells. ROS generation in SH-SY5Y cells was evaluated using CM-H2DCFDA. (A) The generation of ROS in SH-SY5Y cells treated with Aβ (1–10 μM) or CSZ (1, 2.5 μM) for 1 h. (B) The generation of ROS was determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or 10 μM inhibitor of MEK1/2 or p38 MAPK followed by 1 h incubation with Aβ. ROS generation of the control cells was 1455290 ± 70201 Fluorescence intensity. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Incubation, Fluorescence

    Effect of CSZ on Nox activity and the mRNA expression analysis of NOX4 in Aβ-stimulated SH-SY5Y cells. (A) Nox activities were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 1 h incubation with Aβ. Values are expressed as mean + SEM. (B) Nox activities were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Values are expressed as mean + SEM. (C) The mRNA expression analysis of NOX4 in treated SH-SY5Y cells are examined. The effects of respective inhibitor were also examined. Values are expressed as mean + SEM. (D) Reaction cycles-PCR product yield curves of each reaction mixture were plotted. (E) Picture shows the bands obtained in agarose gel electrophoresis for Nox4 and β-actin. Lane1–3: control, Lane4–6: 2.5 μM Aβ, Lane7–9: 2.5 μM CSZ, Lane10–12: 2.5 μM Aβ + 2.5 μM CSZ, Lane13–15: 2.5 μM Aβ + 2.5 μM CSZ + 10 μM PD98059, Lane16–18: 2.5 μM Aβ + 2.5 μM CSZ + 10 μM SB202190. # Compared vs. respective control cells ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effect of CSZ on Nox activity and the mRNA expression analysis of NOX4 in Aβ-stimulated SH-SY5Y cells. (A) Nox activities were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 1 h incubation with Aβ. Values are expressed as mean + SEM. (B) Nox activities were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Values are expressed as mean + SEM. (C) The mRNA expression analysis of NOX4 in treated SH-SY5Y cells are examined. The effects of respective inhibitor were also examined. Values are expressed as mean + SEM. (D) Reaction cycles-PCR product yield curves of each reaction mixture were plotted. (E) Picture shows the bands obtained in agarose gel electrophoresis for Nox4 and β-actin. Lane1–3: control, Lane4–6: 2.5 μM Aβ, Lane7–9: 2.5 μM CSZ, Lane10–12: 2.5 μM Aβ + 2.5 μM CSZ, Lane13–15: 2.5 μM Aβ + 2.5 μM CSZ + 10 μM PD98059, Lane16–18: 2.5 μM Aβ + 2.5 μM CSZ + 10 μM SB202190. # Compared vs. respective control cells ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Activity Assay, Expressing, Incubation, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Effects of CSZ and inhibitors of MEK1/2 and p38 MAPK on Bax, Bcl-2 and cytochrome c in Aβ- timulated SH-SY5Y cells. Levels of Bax and Bcl-2 were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Cells were extracted with a cell lysis buffer and quantitative determination of Bax, Bcl-2 and cytochrome c. (A) Bax (B) Bcl-2 (C) cytochrome c. Values are expressed as mean + SEM # Compared vs. control cells ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effects of CSZ and inhibitors of MEK1/2 and p38 MAPK on Bax, Bcl-2 and cytochrome c in Aβ- timulated SH-SY5Y cells. Levels of Bax and Bcl-2 were determined in SH-SY5Y cells incubated with or without 1 h pretreatment with CSZ or inhibitor of MEK1/2 or p38 MAPK followed by 20 h incubation with Aβ. Cells were extracted with a cell lysis buffer and quantitative determination of Bax, Bcl-2 and cytochrome c. (A) Bax (B) Bcl-2 (C) cytochrome c. Values are expressed as mean + SEM # Compared vs. control cells ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Incubation, Lysis

    Effect of cilostazol (CSZ) on viability and staining with annexin V and Hoechst33342 in amyloid β (Aβ)-stimulated SH-SY5Y cells. We evaluated the effect of Aβ on cell viability using MTT assay and stained with annexin V and Hoechst33342. (A) Cell viability of SH-SY5Y cells treated with Aβ and CSZ for 20 h. (B) The effect of Aβ with or without CSZ on cell viability of SH-SY5Y cells. (C) The effect of Aβ and CSZ with or without the inhibitor of MAPK/ERK kinase1/2 (MEK1/2) (PD98059) or p38 mitogen-activated protein kinases (MAPK) (SB202190) on cell viability of SH-SY5Y cells. Cells were observed under a phase-contrast microscope (D–H) and a fluorescence microscope (I–M) . Images (D,I) untreated SH-SY5Y cells; images (E,J) SH-SY5Y cells treated with Aβ; and images (F,K) : SH-SY5Y cells treated with Aβ + CSZ; images (G,L) SH-SY5Y cells treated with Aβ + CSZ + inhibitor of MEK1/2; images (H,M) SH-SY5Y cells treated with Aβ + CSZ + inhibitor of p38 MAPK. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Cilostazol Suppresses Aβ-induced Neurotoxicity in SH-SY5Y Cells through Inhibition of Oxidative Stress and MAPK Signaling Pathway

    doi: 10.3389/fnagi.2017.00337

    Figure Lengend Snippet: Effect of cilostazol (CSZ) on viability and staining with annexin V and Hoechst33342 in amyloid β (Aβ)-stimulated SH-SY5Y cells. We evaluated the effect of Aβ on cell viability using MTT assay and stained with annexin V and Hoechst33342. (A) Cell viability of SH-SY5Y cells treated with Aβ and CSZ for 20 h. (B) The effect of Aβ with or without CSZ on cell viability of SH-SY5Y cells. (C) The effect of Aβ and CSZ with or without the inhibitor of MAPK/ERK kinase1/2 (MEK1/2) (PD98059) or p38 mitogen-activated protein kinases (MAPK) (SB202190) on cell viability of SH-SY5Y cells. Cells were observed under a phase-contrast microscope (D–H) and a fluorescence microscope (I–M) . Images (D,I) untreated SH-SY5Y cells; images (E,J) SH-SY5Y cells treated with Aβ; and images (F,K) : SH-SY5Y cells treated with Aβ + CSZ; images (G,L) SH-SY5Y cells treated with Aβ + CSZ + inhibitor of MEK1/2; images (H,M) SH-SY5Y cells treated with Aβ + CSZ + inhibitor of p38 MAPK. Values are expressed as mean + SEM. # Compared vs. respective controls ( p

    Article Snippet: For the quantitative determination of Bax and Bcl-2 in SH-SY5Y cells lysates, Bax human EIA kit (Enzo Life Sciences Int’l, Inc., NY, USA) and Human Bcl-2 Platinum ELISA kit (eBioscience, Vienna, Austria) were used, respectively.

    Techniques: Staining, MTT Assay, Microscopy, Fluorescence

    The effect of 3′ overhang on RISC function in Dicer kd, Ago2 kd and TRBP kd cells. Efficiencies of target downregulation by four variants of EGFPS1A siRNA ( Figure 1 ) were evaluated using Dual Luciferase assays with siDicer, siAgo2 or siTRBP treated HEK293 cells. To determine effects of Dicer, Ago2 or TRBP knockdowns on the silencing activity of the siEGFPS1A variants described in Figure 1 , HEK293 cells were treated with 40 nM siDicer, siAgo2 or siTRBP for 2 days prior to the co-transfection. For the second round of transfection, Dicer kd, Ago2 kd or TRBP kd cells were transfected with the psi-EGFP-S1 sense reporter and 200 pM siEGFPS1A variants. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from four co-transfections).

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: The effect of 3′ overhang on RISC function in Dicer kd, Ago2 kd and TRBP kd cells. Efficiencies of target downregulation by four variants of EGFPS1A siRNA ( Figure 1 ) were evaluated using Dual Luciferase assays with siDicer, siAgo2 or siTRBP treated HEK293 cells. To determine effects of Dicer, Ago2 or TRBP knockdowns on the silencing activity of the siEGFPS1A variants described in Figure 1 , HEK293 cells were treated with 40 nM siDicer, siAgo2 or siTRBP for 2 days prior to the co-transfection. For the second round of transfection, Dicer kd, Ago2 kd or TRBP kd cells were transfected with the psi-EGFP-S1 sense reporter and 200 pM siEGFPS1A variants. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from four co-transfections).

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Luciferase, Activity Assay, Cotransfection, Transfection, Expressing

    The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. ( c ) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. ( c ) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Negative Control, Incubation, Luciferase, Modification, Expressing, Transfection

    Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. ( a ) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I ( 40 ). Upper strand is sense; bottom strand is antisense. ( b ) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1 . The assays were conducted multiple times with similar results, and a representative gel is shown here. ( c ) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1 ). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. ( a ) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I ( 40 ). Upper strand is sense; bottom strand is antisense. ( b ) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1 . The assays were conducted multiple times with similar results, and a representative gel is shown here. ( c ) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1 ). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Transfection, Plasmid Preparation, Luciferase, Reporter Assay

    Analysis of RISC components required in high-molecular-complex formation. Gel-shift assays. EGFPS1A duplex was incubated with HEK293 cell extracts immunodepleted of Dicer (Id Dicer), TRBP (Id TRBP), Dicer and TRBP (Id Dicer/TRBP), Ago2 (Id Ago2) or Ago2 and TRBP (Id Ago2/TRBP). Evaluation of immunodepletion of RISC proteins with western blot analyses was shown on the right. Of Id cell extracts, 150 µg were loaded on a 8% SDS–PAGE gel. Pre-immune serum was used as an immunodepletion control.

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: Analysis of RISC components required in high-molecular-complex formation. Gel-shift assays. EGFPS1A duplex was incubated with HEK293 cell extracts immunodepleted of Dicer (Id Dicer), TRBP (Id TRBP), Dicer and TRBP (Id Dicer/TRBP), Ago2 (Id Ago2) or Ago2 and TRBP (Id Ago2/TRBP). Evaluation of immunodepletion of RISC proteins with western blot analyses was shown on the right. Of Id cell extracts, 150 µg were loaded on a 8% SDS–PAGE gel. Pre-immune serum was used as an immunodepletion control.

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Western Blot, SDS Page

    Analysis of siRNA–RISC complex formation. ( a ) Reconstitution of the siRNA complex with recombinant proteins. Id Dicer (left) or Id TRBP (right) cell extracts were mixed with 2 µg of recombinant Dicer, 80 ng of recombinant TRBP or both proteins to rescue complex formation. ( b and c ) Percent-bound-reconstituted siRNA complexes were calculated relative to the HEK293 WCE which was set as 100%.

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: Analysis of siRNA–RISC complex formation. ( a ) Reconstitution of the siRNA complex with recombinant proteins. Id Dicer (left) or Id TRBP (right) cell extracts were mixed with 2 µg of recombinant Dicer, 80 ng of recombinant TRBP or both proteins to rescue complex formation. ( b and c ) Percent-bound-reconstituted siRNA complexes were calculated relative to the HEK293 WCE which was set as 100%.

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Recombinant

    Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Incubation, Western Blot, SDS Page

    Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A ( a ) and EGFPS1B ( b ) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC 50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.

    Journal: Nucleic Acids Research

    Article Title: A role for human Dicer in pre-RISC loading of siRNAs

    doi: 10.1093/nar/gkq846

    Figure Lengend Snippet: Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A ( a ) and EGFPS1B ( b ) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC 50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.

    Article Snippet: HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega).

    Techniques: Selection, Transfection, Luciferase, Expressing, Blocking Assay

    PGRMC2 interacts with ALADIN determined by IP-western and reciprocal IP- w estern assays. (A) Whole cell lysates of GFP-ALADIN, PGRMC2-GFP- and GFP- (as negative control) expressing NCI-H295R cells were used and GFP pulldown performed followed by western blot with indicated antibodies. PGRMC2 (24 kDa, arrow) could be detected after GFP-ALADIN (86 kDa) pulldown. ALADIN (59 kDa, arrow) and PGRMC2 could be both detected after PGRMC2-GFP (51 kDa) pulldown. GFP (27 kDa) was ascertained after GFP control pulldown but the control remained empty for PGRMC2 and ALADIN. (B) Whole cell lysates of NCI-H295R cells were used for ALADIN and PGRMC2 pulldown. Normal mouse (m IgG) and rabbit IgGs (Rb IgG) served as negative control. Western blot was performed with indicated antibodies. Non-bound (NB) IP fractions are also shown for each pulldown and bound IP eluates are separated by one lane each from the specific controls to eliminate false positive detection. PGRMC2 (arrow) precipitated in endogenous ALADIN pulldown. Control m IgG pulldown remained empty. ALADIN (arrow) reciprocally precipitated after PGRMC2 pulldown. Control Rb IgG pulldown remained empty showing a cross-reactive band a few kDa higher than ALADIN.

    Journal: Biology Open

    Article Title: Identification of a novel putative interaction partner of the nucleoporin ALADIN

    doi: 10.1242/bio.021162

    Figure Lengend Snippet: PGRMC2 interacts with ALADIN determined by IP-western and reciprocal IP- w estern assays. (A) Whole cell lysates of GFP-ALADIN, PGRMC2-GFP- and GFP- (as negative control) expressing NCI-H295R cells were used and GFP pulldown performed followed by western blot with indicated antibodies. PGRMC2 (24 kDa, arrow) could be detected after GFP-ALADIN (86 kDa) pulldown. ALADIN (59 kDa, arrow) and PGRMC2 could be both detected after PGRMC2-GFP (51 kDa) pulldown. GFP (27 kDa) was ascertained after GFP control pulldown but the control remained empty for PGRMC2 and ALADIN. (B) Whole cell lysates of NCI-H295R cells were used for ALADIN and PGRMC2 pulldown. Normal mouse (m IgG) and rabbit IgGs (Rb IgG) served as negative control. Western blot was performed with indicated antibodies. Non-bound (NB) IP fractions are also shown for each pulldown and bound IP eluates are separated by one lane each from the specific controls to eliminate false positive detection. PGRMC2 (arrow) precipitated in endogenous ALADIN pulldown. Control m IgG pulldown remained empty. ALADIN (arrow) reciprocally precipitated after PGRMC2 pulldown. Control Rb IgG pulldown remained empty showing a cross-reactive band a few kDa higher than ALADIN.

    Article Snippet: For co-IP of ALADIN or PGRMC2 lysates from NCI-H295R cells and Protein G UltraLink resin sepharose beads (Pierce, Thermo Scientific, Fischer Scientific, Schwerte, Germany) were used.

    Techniques: Western Blot, Negative Control, Expressing

    Pgrmc2 has a sexual dimorphic role in mice and ALADIN KO in female mice leads to an alteration in PGRMC2. (A) Total RNA was isolated from dissected adrenals and gonads of WT and Aaas KO mice. *P

    Journal: Biology Open

    Article Title: Identification of a novel putative interaction partner of the nucleoporin ALADIN

    doi: 10.1242/bio.021162

    Figure Lengend Snippet: Pgrmc2 has a sexual dimorphic role in mice and ALADIN KO in female mice leads to an alteration in PGRMC2. (A) Total RNA was isolated from dissected adrenals and gonads of WT and Aaas KO mice. *P

    Article Snippet: For co-IP of ALADIN or PGRMC2 lysates from NCI-H295R cells and Protein G UltraLink resin sepharose beads (Pierce, Thermo Scientific, Fischer Scientific, Schwerte, Germany) were used.

    Techniques: Mouse Assay, Gene Knockout, Isolation

    ALADIN and PGRMC2 localise to the perinuclear space in human adrenocortical carcinoma cells. NCI-H295R/GFP-ALADIN, NCI-H295R/PGRMC2-GFP, NCI-H295R and NCI-H295R/GFP cells were stained with anti-ALADIN (red), anti-PGRMC2 [red (F-3) and green (HPA041172)], anti-NPC proteins (mAb414) (red) and DAPI (blue). Scale bars: 11 µm (NCI-H295R/GFP-ALADIN, NCI-H295R), 12 µm (NCI-H295R/PGRMC2-GFP) and 16 µm (NCI-H295R/GFP).

    Journal: Biology Open

    Article Title: Identification of a novel putative interaction partner of the nucleoporin ALADIN

    doi: 10.1242/bio.021162

    Figure Lengend Snippet: ALADIN and PGRMC2 localise to the perinuclear space in human adrenocortical carcinoma cells. NCI-H295R/GFP-ALADIN, NCI-H295R/PGRMC2-GFP, NCI-H295R and NCI-H295R/GFP cells were stained with anti-ALADIN (red), anti-PGRMC2 [red (F-3) and green (HPA041172)], anti-NPC proteins (mAb414) (red) and DAPI (blue). Scale bars: 11 µm (NCI-H295R/GFP-ALADIN, NCI-H295R), 12 µm (NCI-H295R/PGRMC2-GFP) and 16 µm (NCI-H295R/GFP).

    Article Snippet: For co-IP of ALADIN or PGRMC2 lysates from NCI-H295R cells and Protein G UltraLink resin sepharose beads (Pierce, Thermo Scientific, Fischer Scientific, Schwerte, Germany) were used.

    Techniques: Staining

    Depletion of ALADIN affects PGRMC2 localisation at the nuclear envelope/perinuclear ER in human skin fibroblasts. NCI-H295R1-TR/ AAAS knock-down, NCI-H295R1-TR/Scrambled shRNA, NCI-H295R1-TR cells and skin fibroblasts of a triple A patient (homozygous mutation in Exon 9, c.884G > A, p.Trp295X) and of an anonymised control were stained with mouse anti-ALADIN (green), mouse anti-PGRMC2 (green) and DAPI (blue). Scale bars for anti-ALADIN staining (upper panels): 38 µm (NCI-H295R1-TR/ AAAS knock-down), 24 µm (NCI-H295R1-TR/Scrambled shRNA), 32 µm (NCI-H295R1-TR cells) and 38 µm (skin fibroblasts). All scale bars for anti-PGRMC2 staining: 24 µm. NCI-H295R1-TR cells with AAAS knock-down or scrambled shRNA were induced as described previously ( Jühlen et al., 2015 ).

    Journal: Biology Open

    Article Title: Identification of a novel putative interaction partner of the nucleoporin ALADIN

    doi: 10.1242/bio.021162

    Figure Lengend Snippet: Depletion of ALADIN affects PGRMC2 localisation at the nuclear envelope/perinuclear ER in human skin fibroblasts. NCI-H295R1-TR/ AAAS knock-down, NCI-H295R1-TR/Scrambled shRNA, NCI-H295R1-TR cells and skin fibroblasts of a triple A patient (homozygous mutation in Exon 9, c.884G > A, p.Trp295X) and of an anonymised control were stained with mouse anti-ALADIN (green), mouse anti-PGRMC2 (green) and DAPI (blue). Scale bars for anti-ALADIN staining (upper panels): 38 µm (NCI-H295R1-TR/ AAAS knock-down), 24 µm (NCI-H295R1-TR/Scrambled shRNA), 32 µm (NCI-H295R1-TR cells) and 38 µm (skin fibroblasts). All scale bars for anti-PGRMC2 staining: 24 µm. NCI-H295R1-TR cells with AAAS knock-down or scrambled shRNA were induced as described previously ( Jühlen et al., 2015 ).

    Article Snippet: For co-IP of ALADIN or PGRMC2 lysates from NCI-H295R cells and Protein G UltraLink resin sepharose beads (Pierce, Thermo Scientific, Fischer Scientific, Schwerte, Germany) were used.

    Techniques: shRNA, Mutagenesis, Staining

    PGRMC2 was identified in mass spectrometry after GFP-(ALADIN) pulldown. Identified exclusive unique peptides (yellow) (number of different amino acid sequences, regardless of any modification, that are associated only with this protein) of ALADIN and progesterone receptor membrane component 2 (PGRMC2) are highlighted. Also for PGRMC2, relative intensities of annotated spectra are shown after GFP(-ALADIN) co-IP of whole cell lysates of GFP-ALADIN expressing NCI-H295R cells.

    Journal: Biology Open

    Article Title: Identification of a novel putative interaction partner of the nucleoporin ALADIN

    doi: 10.1242/bio.021162

    Figure Lengend Snippet: PGRMC2 was identified in mass spectrometry after GFP-(ALADIN) pulldown. Identified exclusive unique peptides (yellow) (number of different amino acid sequences, regardless of any modification, that are associated only with this protein) of ALADIN and progesterone receptor membrane component 2 (PGRMC2) are highlighted. Also for PGRMC2, relative intensities of annotated spectra are shown after GFP(-ALADIN) co-IP of whole cell lysates of GFP-ALADIN expressing NCI-H295R cells.

    Article Snippet: For co-IP of ALADIN or PGRMC2 lysates from NCI-H295R cells and Protein G UltraLink resin sepharose beads (Pierce, Thermo Scientific, Fischer Scientific, Schwerte, Germany) were used.

    Techniques: Mass Spectrometry, Modification, Co-Immunoprecipitation Assay, Expressing

    Extent of G 2 arrest is enhanced in E4-17/16K-expressing cells by overexpression of Wee1. (A) Western blot analysis of Wee1 protein levels in HeLa cells either mock transfected (−) or transfected (+) with Wee1 expression plasmid. Fluorescence

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Extent of G 2 arrest is enhanced in E4-17/16K-expressing cells by overexpression of Wee1. (A) Western blot analysis of Wee1 protein levels in HeLa cells either mock transfected (−) or transfected (+) with Wee1 expression plasmid. Fluorescence

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Expressing, Over Expression, Western Blot, Transfection, Plasmid Preparation, Fluorescence

    Depletion of Wee1 by siRNA inhibits G 2 arrest induced by E4-17/16K protein expression. (A) Western blot analysis of the Wee1 protein in HeLa cells either mock transfected or transfected with siRNAs specific for Wee1 (siWee1) or Luciferase (siLuciferase),

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Depletion of Wee1 by siRNA inhibits G 2 arrest induced by E4-17/16K protein expression. (A) Western blot analysis of the Wee1 protein in HeLa cells either mock transfected or transfected with siRNAs specific for Wee1 (siWee1) or Luciferase (siLuciferase),

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Expressing, Western Blot, Transfection, Luciferase

    Inhibition of G 2 -to-M transition following coexpression of E4-17/16K proteins correlates with increased levels of phosphorylated cdk1. (A) cdk1 protein expression profiles in HeLa cells either mock infected or infected with rAds for 72 h. HeLa cells treated

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Inhibition of G 2 -to-M transition following coexpression of E4-17/16K proteins correlates with increased levels of phosphorylated cdk1. (A) cdk1 protein expression profiles in HeLa cells either mock infected or infected with rAds for 72 h. HeLa cells treated

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Inhibition, Expressing, Infection

    E4-17K and E4-16K form a heteromeric complex. Western blot analysis (MAb 4.37) of HA tag immune complexes isolated from HeLa cells transfected with a 17K-HA or 16K expression plasmid or cells cotransfected with both plasmids (17K-HA plus 16K). Control

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: E4-17K and E4-16K form a heteromeric complex. Western blot analysis (MAb 4.37) of HA tag immune complexes isolated from HeLa cells transfected with a 17K-HA or 16K expression plasmid or cells cotransfected with both plasmids (17K-HA plus 16K). Control

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Western Blot, Hemagglutination Assay, Isolation, Transfection, Expressing, Plasmid Preparation

    Differences in the level of cyclin B1 protein and cdk1 activity between HeLa cells arrested in G 2 by E4-16K expression or combined expression of E4-17K and E4-16K. (A) Cell cycle analysis of HeLa cells either mock infected (MI) or infected with rAds expressing

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Differences in the level of cyclin B1 protein and cdk1 activity between HeLa cells arrested in G 2 by E4-16K expression or combined expression of E4-17K and E4-16K. (A) Cell cycle analysis of HeLa cells either mock infected (MI) or infected with rAds expressing

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Activity Assay, Expressing, Cell Cycle Assay, Infection

    Level of Wee1 kinase is stabilized in HeLa cells arrested in G 2 following coexpression of the E4-17K and E4-16K proteins. Wee1 protein expression in synchronous HeLa cells infected with rAds expressing E4-17K or E4-16K or coinfected with both viruses

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Level of Wee1 kinase is stabilized in HeLa cells arrested in G 2 following coexpression of the E4-17K and E4-16K proteins. Wee1 protein expression in synchronous HeLa cells infected with rAds expressing E4-17K or E4-16K or coinfected with both viruses

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Expressing, Infection

    Inhibition of PP2A abrogates inactivation of cdk1 activity in cells coexpressing E4-17/16K proteins. HeLa cells either mock infected or infected with rAds expressing the different E4 proteins were treated with 10 μM okadaic acid (+OA)

    Journal:

    Article Title: Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

    doi: 10.1128/JVI.00196-06

    Figure Lengend Snippet: Inhibition of PP2A abrogates inactivation of cdk1 activity in cells coexpressing E4-17/16K proteins. HeLa cells either mock infected or infected with rAds expressing the different E4 proteins were treated with 10 μM okadaic acid (+OA)

    Article Snippet: Because inhibitory phosphates are removed from cdk1 by cdc25c, we examined the possibility that cdc25c was inactive in E4-17K/16K cells by immunoblotting the HeLa cell lysates with an antibody that recognizes the inactive form of the phosphatase (rabbit antibody that recognizes phosphorylation of Ser216 on cdc25c (no. 9528; Cell Signaling Inc.).

    Techniques: Inhibition, Activity Assay, Infection, Expressing

    Differences in lipid profiling between prom1-exo (MV) and parental FEMX-I cells (FEMX). An automated ESI-tandem mass spectrometry approach was used for lipid profiling. Averages of three to five determinations for each sample group were calculated. Red, lipid species over-expressed in prom1-exo; blue, lipid species over-expressed in parental FEMX-I cells. *, p

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Differences in lipid profiling between prom1-exo (MV) and parental FEMX-I cells (FEMX). An automated ESI-tandem mass spectrometry approach was used for lipid profiling. Averages of three to five determinations for each sample group were calculated. Red, lipid species over-expressed in prom1-exo; blue, lipid species over-expressed in parental FEMX-I cells. *, p

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques: Mass Spectrometry

    Co - culture of MSC and FEMX-I cells shows uptake of prominin-1 by MSC. MSC and FEMX-I cells were cultured for 24 h at 1:5 ratio. After fixation and permeabilization, expression of prominin-1 was analyzed by immunofluorescence employing phycoerythrin-conjugated AC133 anti-prominin-1 antibody. Since MSC do not express prominin-1, there could be no interference from an endogenous MSC prominin-1 pool. Insets in the upper panels were enlarged in the lower panels. Arrows indicate some areas of prominin-1 positivity inside a MSC. Red, prominin-1; blue, DAPI. Bars, 25 μm.

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Co - culture of MSC and FEMX-I cells shows uptake of prominin-1 by MSC. MSC and FEMX-I cells were cultured for 24 h at 1:5 ratio. After fixation and permeabilization, expression of prominin-1 was analyzed by immunofluorescence employing phycoerythrin-conjugated AC133 anti-prominin-1 antibody. Since MSC do not express prominin-1, there could be no interference from an endogenous MSC prominin-1 pool. Insets in the upper panels were enlarged in the lower panels. Arrows indicate some areas of prominin-1 positivity inside a MSC. Red, prominin-1; blue, DAPI. Bars, 25 μm.

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques: Co-Culture Assay, Cell Culture, Expressing, Immunofluorescence

    Enrichment of prominin-1 and alix in prom1-exo. Immunoblotting analysis of total cell lysates, microvesicles (MVs), and prom1-exo from FEMX-I cells. 1 and 10 μg of total proteins were loaded per lane for total cell lysates and MVs and 1 μg for prom1-exo, and analyzed as described under Experimental Procedures .

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Enrichment of prominin-1 and alix in prom1-exo. Immunoblotting analysis of total cell lysates, microvesicles (MVs), and prom1-exo from FEMX-I cells. 1 and 10 μg of total proteins were loaded per lane for total cell lysates and MVs and 1 μg for prom1-exo, and analyzed as described under Experimental Procedures .

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques:

    Co-localization of prominin-1 with CD29 in FEMX-I cells. Insets in the upper panels were enlarged in the lower panels. Arrows represent areas of peri-nuclear co-localization of prominin-1 and CD29. Prominin-1, red. CD29, green; DAPI, blue. Bars, 25 μm.

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Co-localization of prominin-1 with CD29 in FEMX-I cells. Insets in the upper panels were enlarged in the lower panels. Arrows represent areas of peri-nuclear co-localization of prominin-1 and CD29. Prominin-1, red. CD29, green; DAPI, blue. Bars, 25 μm.

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques:

    Different membrane lipid distribution between parental FEMX-I cells and prom1-exo. An automated ESI-tandem mass spectrometry approach was used. The lipid extracts from cells and microvesicles were dissolved in 1 ml chloroform. An aliquot of 50 μl of each extract in chloroform was used for each analysis. To correct for chemical or instrumental noise in the samples, the molar amount of each lipid metabolite detected in the “internal standards only” spectra was subtracted from the molar amount of each metabolite calculated in each set of sample spectra. The data from each “internal standards only” set of spectra was used to correct the data from the following 10 samples. Finally, the data were corrected for the fraction of the sample analyzed and normalized to the mg protein to produce data in the units nmol/mg. Data are presented as percent of total lipids analyzed. *, p

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Different membrane lipid distribution between parental FEMX-I cells and prom1-exo. An automated ESI-tandem mass spectrometry approach was used. The lipid extracts from cells and microvesicles were dissolved in 1 ml chloroform. An aliquot of 50 μl of each extract in chloroform was used for each analysis. To correct for chemical or instrumental noise in the samples, the molar amount of each lipid metabolite detected in the “internal standards only” spectra was subtracted from the molar amount of each metabolite calculated in each set of sample spectra. The data from each “internal standards only” set of spectra was used to correct the data from the following 10 samples. Finally, the data were corrected for the fraction of the sample analyzed and normalized to the mg protein to produce data in the units nmol/mg. Data are presented as percent of total lipids analyzed. *, p

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques: Mass Spectrometry

    Uptake of prom1-exo by FEMX-I and MSC. A . Cells were incubated for 3 h with PKH67-labeled green fluorescent prom1-exo, fixed and stained with phycoerythrin-conjugated AC133 anti-prominin-1 antibody. Arrows represent areas of co-localization of green PKH67 fluorescence and red fluorescent anti-prominin-1 antibodies. Since MSC do not express prominin-1, there could be no interference from an endogenous MSC prominin-1 pool. Bars, 25 μm. B . MSC were incubated for 3 h with prom1-exo prepared from FEMX-I cells transiently transfected with a prominin-1-GFP fusion plasmid.

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Uptake of prom1-exo by FEMX-I and MSC. A . Cells were incubated for 3 h with PKH67-labeled green fluorescent prom1-exo, fixed and stained with phycoerythrin-conjugated AC133 anti-prominin-1 antibody. Arrows represent areas of co-localization of green PKH67 fluorescence and red fluorescent anti-prominin-1 antibodies. Since MSC do not express prominin-1, there could be no interference from an endogenous MSC prominin-1 pool. Bars, 25 μm. B . MSC were incubated for 3 h with prom1-exo prepared from FEMX-I cells transiently transfected with a prominin-1-GFP fusion plasmid.

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques: Incubation, Labeling, Staining, Fluorescence, Transfection, Plasmid Preparation

    Isolation and characterization of prom1-exo from FEMX-I cells. A . Scheme of isolation of “classical” microvesicles by differential centrifugation and of prom1-exo by a combination of differential centrifugation, filtration and immuno-magnetic separation. B . Microvesicle tracking analysis shows size distribution of a “classical” ultracentrifugation-based preparation of microvesicles and a prominin-1-based immunomagnetic preparation (prom1-exo), both from serum-free culture medium of the human FEMX-I metastatic melanoma cell line. Both preparations were stained with the membrane dye PKH67 and fluorescence analyzed by a 488 nm laser. Nanotracking analysis gives mean peak intensities of 80 and 120 nm for microvesicles and 90 nm for exosomes, respectively. The persistent binding of magnetic beads (50 nm) to the prominin-1 microvesicles resulted in an over-estimation of their size distribution.

    Journal: Molecular Cancer

    Article Title: Biochemical and biological characterization of exosomes containing prominin-1/CD133

    doi: 10.1186/1476-4598-12-62

    Figure Lengend Snippet: Isolation and characterization of prom1-exo from FEMX-I cells. A . Scheme of isolation of “classical” microvesicles by differential centrifugation and of prom1-exo by a combination of differential centrifugation, filtration and immuno-magnetic separation. B . Microvesicle tracking analysis shows size distribution of a “classical” ultracentrifugation-based preparation of microvesicles and a prominin-1-based immunomagnetic preparation (prom1-exo), both from serum-free culture medium of the human FEMX-I metastatic melanoma cell line. Both preparations were stained with the membrane dye PKH67 and fluorescence analyzed by a 488 nm laser. Nanotracking analysis gives mean peak intensities of 80 and 120 nm for microvesicles and 90 nm for exosomes, respectively. The persistent binding of magnetic beads (50 nm) to the prominin-1 microvesicles resulted in an over-estimation of their size distribution.

    Article Snippet: Aliquots of microvesicles, exosomes and FEMX-I total cell lysates containing 1–10 μg of protein were mixed 1:1 with SDS sample buffer (NuSep, Bogart, GA) containing 2% 2-mercaptoethanol, boiled for 5 min, and loaded onto a 8% Tris/Glycine/SDS gel.

    Techniques: Isolation, Centrifugation, Filtration, Staining, Fluorescence, Binding Assay, Magnetic Beads

    Phosphopeptide microarray analysis of human cardiomyocyte progenitor cells showing detection of multiple proteins (GRB2 and SRC) on the same phosphoprotein-protein interaction complexes. The red spots refer to the presence of GRB2 protein (Rabbit Anti GRB2 with Rabbit anti-IgG conjugated with Alexa 647); The green spots refer to SRC (Mouse Anti SRC with Mouse anti-IgG conjugated with Alexa 594); The yellow spots is the results of merge of GRB2 and SRC images together showing the presence of GRB2 and SRC on the same phosphopeptide-protein complex.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Phosphopeptide microarray analysis of human cardiomyocyte progenitor cells showing detection of multiple proteins (GRB2 and SRC) on the same phosphoprotein-protein interaction complexes. The red spots refer to the presence of GRB2 protein (Rabbit Anti GRB2 with Rabbit anti-IgG conjugated with Alexa 647); The green spots refer to SRC (Mouse Anti SRC with Mouse anti-IgG conjugated with Alexa 594); The yellow spots is the results of merge of GRB2 and SRC images together showing the presence of GRB2 and SRC on the same phosphopeptide-protein complex.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Microarray

    Details of recombinant protein binding assay on peptide microarray. 2(a) Flow diagram showing the steps of recombinant protein binding assay on the pY peptide microarray synthesized on the chip surface. The recombinant protein containing an affinity tag (such as HIS, or GST) was applied to the microfluidic chip to be in contact with the pY peptide probes for about 1 hour. The specific interaction (binding) occurs between the protein and the pY peptide probe peptides based on the affinity of SH2 domain with phosphotyrosine residues on specific protein motifs. The binding was recognized by primary antibody (such as anti-HIS or anti-GST). Detection of the binding is through fluorescence dye (Hylite)-conjugated primary antibody. 2(b) Recombinant protein binding on peptide array: Quality of binding image, data processing and analysis: The chip contained 3986 peptides synthesized in situ on a microfluidic chip surface containing 3,968 picoliter reaction wells with 1,227 peptides were tyrosine phosphorylated (pY-peptides) and 1227 corresponding control peptides (A-peptides) with technical controls for monitoring synthesis quality and protein binding assays. A . Binding image of GST-Grb2 SH2 domain on phosphopeptide chip: The array was probed with GST-tagged Grb2-SH2 domain and detected by antiGST-Hylite 647 conjugated antibody. Signals from proteins bound to phosphopeptides compared and respective control peptides wells were scored. B. Representative image of a small region from panel A. Peptide sequences (pY-peptides and A-peptides) are provided on either side of the image panel with the pY sequences marked in red and A-peptide sequences in black. The pY-peptide and corresponding A-peptide were synthesized at the two adjacent positions in the same column for conveniently visualizing pY specific binding. C . Histogram of the Grb2 binding (log 10 scale) showing the distribution of binding intensities ranging from 500 to 60000 with a mean intensity around 1500. D . Histogram plot of the spot to spot CV (covariance of three replicate peptide-probes on chip) distribution of Grb2 binding: 3,695 (93%) spots have CVs less than 0.25, showing both peptide synthesis and Grb2 binding are uniform. E . Bar graph plot of the detected intensities of Grb2 SH2 domain binding shown as blue ( pY -peptide probe signals) or brown ( A -peptide probe signals). Grb2 SH2 domain binding affinity varied from 5 to 30 folds compared to control peptides. 2(c) Comparison of peptide microarray SH2 domain binding motif consensus of this study (BTK, Grb2, Src and ZAP70) with those of previously published in vitro peptide binding results: Binding patterns and consensus sequences of four SH2 domain containing proteins (BTK, Grb2, Src and ZAP70). A. Binding images of four SH2 domains on the same region of four independent phosphopeptide chips. B. Heat map showing differential binding intensities (on Z value scale) of 4 SH2 containing proteins to related PPEPs on the chips. The pY probes with Z values ranked from 1.0–3.0 were plotted. C. Consensus sequences of binding sequences of 4 SH2 containing proteins. The consensus sequences were aligned by using WebLogo (version 2.8.2 http://weblogo.berkeley.edu/logo.cgi/ ). For each SH2 domain, the binding PPEPs with Z values greater than 1 were selected. The number of high confidence peptide probes selected to BTK, Grb2, Src and ZAP70 are 35, 133, 86 and 45, respectively.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Details of recombinant protein binding assay on peptide microarray. 2(a) Flow diagram showing the steps of recombinant protein binding assay on the pY peptide microarray synthesized on the chip surface. The recombinant protein containing an affinity tag (such as HIS, or GST) was applied to the microfluidic chip to be in contact with the pY peptide probes for about 1 hour. The specific interaction (binding) occurs between the protein and the pY peptide probe peptides based on the affinity of SH2 domain with phosphotyrosine residues on specific protein motifs. The binding was recognized by primary antibody (such as anti-HIS or anti-GST). Detection of the binding is through fluorescence dye (Hylite)-conjugated primary antibody. 2(b) Recombinant protein binding on peptide array: Quality of binding image, data processing and analysis: The chip contained 3986 peptides synthesized in situ on a microfluidic chip surface containing 3,968 picoliter reaction wells with 1,227 peptides were tyrosine phosphorylated (pY-peptides) and 1227 corresponding control peptides (A-peptides) with technical controls for monitoring synthesis quality and protein binding assays. A . Binding image of GST-Grb2 SH2 domain on phosphopeptide chip: The array was probed with GST-tagged Grb2-SH2 domain and detected by antiGST-Hylite 647 conjugated antibody. Signals from proteins bound to phosphopeptides compared and respective control peptides wells were scored. B. Representative image of a small region from panel A. Peptide sequences (pY-peptides and A-peptides) are provided on either side of the image panel with the pY sequences marked in red and A-peptide sequences in black. The pY-peptide and corresponding A-peptide were synthesized at the two adjacent positions in the same column for conveniently visualizing pY specific binding. C . Histogram of the Grb2 binding (log 10 scale) showing the distribution of binding intensities ranging from 500 to 60000 with a mean intensity around 1500. D . Histogram plot of the spot to spot CV (covariance of three replicate peptide-probes on chip) distribution of Grb2 binding: 3,695 (93%) spots have CVs less than 0.25, showing both peptide synthesis and Grb2 binding are uniform. E . Bar graph plot of the detected intensities of Grb2 SH2 domain binding shown as blue ( pY -peptide probe signals) or brown ( A -peptide probe signals). Grb2 SH2 domain binding affinity varied from 5 to 30 folds compared to control peptides. 2(c) Comparison of peptide microarray SH2 domain binding motif consensus of this study (BTK, Grb2, Src and ZAP70) with those of previously published in vitro peptide binding results: Binding patterns and consensus sequences of four SH2 domain containing proteins (BTK, Grb2, Src and ZAP70). A. Binding images of four SH2 domains on the same region of four independent phosphopeptide chips. B. Heat map showing differential binding intensities (on Z value scale) of 4 SH2 containing proteins to related PPEPs on the chips. The pY probes with Z values ranked from 1.0–3.0 were plotted. C. Consensus sequences of binding sequences of 4 SH2 containing proteins. The consensus sequences were aligned by using WebLogo (version 2.8.2 http://weblogo.berkeley.edu/logo.cgi/ ). For each SH2 domain, the binding PPEPs with Z values greater than 1 were selected. The number of high confidence peptide probes selected to BTK, Grb2, Src and ZAP70 are 35, 133, 86 and 45, respectively.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Recombinant, Protein Binding, Peptide Microarray, Flow Cytometry, Synthesized, Chromatin Immunoprecipitation, Binding Assay, Fluorescence, In Situ, In Vitro

    Graphical representation of dynamics of specific GRB2-tyrosine phosphopeptide interactions across all the four cell lines (MCF10A, MCF7, T47D and MDA-MB231). 7a. Graph showing upregulation of GRB2 association with the MET receptor and FAK kinase. MCF7 and T47D are tumor cells and are ER positive while MCF10A and MDA-MB231 are ER negative. The observed decrease of GRB2 signals is consistent with previous studies reported. GRB2 interactions are significantly downregulated in metastatic cells (MDA-MB231). 7b. Graph showing upregulation of GRB2 association with probes representing adaptor proteins IRS1 and SHC1 in R positive breast cancer cells. Based on reported literature, interactions of GRB2 with IRS1 pY motifs reveal potential cross talk between RTK and ER pathways. 7c. Graph showing upregulation of GRB2 association with adaptor protein pY motifs in immune signaling (B cell and T cell) pathways: These interactions may indicate potential signaling events in tumor cells to overcome cellular immune defense mechanisms of the host cells. 7d. Graph showing upregulation of GRB2 association with specific pY motifs on PTPN11 (SHP-2) protein: Down regulation of motif pY279 association compared to that of pY584 is with downregulation of ERK activation in tumor cells and suggesting alternate RTK pathways through pY584 for ERK activation. Each pY probe intensity value shown in the graph is an average of three replicates on the chip.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Graphical representation of dynamics of specific GRB2-tyrosine phosphopeptide interactions across all the four cell lines (MCF10A, MCF7, T47D and MDA-MB231). 7a. Graph showing upregulation of GRB2 association with the MET receptor and FAK kinase. MCF7 and T47D are tumor cells and are ER positive while MCF10A and MDA-MB231 are ER negative. The observed decrease of GRB2 signals is consistent with previous studies reported. GRB2 interactions are significantly downregulated in metastatic cells (MDA-MB231). 7b. Graph showing upregulation of GRB2 association with probes representing adaptor proteins IRS1 and SHC1 in R positive breast cancer cells. Based on reported literature, interactions of GRB2 with IRS1 pY motifs reveal potential cross talk between RTK and ER pathways. 7c. Graph showing upregulation of GRB2 association with adaptor protein pY motifs in immune signaling (B cell and T cell) pathways: These interactions may indicate potential signaling events in tumor cells to overcome cellular immune defense mechanisms of the host cells. 7d. Graph showing upregulation of GRB2 association with specific pY motifs on PTPN11 (SHP-2) protein: Down regulation of motif pY279 association compared to that of pY584 is with downregulation of ERK activation in tumor cells and suggesting alternate RTK pathways through pY584 for ERK activation. Each pY probe intensity value shown in the graph is an average of three replicates on the chip.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Multiple Displacement Amplification, Activation Assay, Chromatin Immunoprecipitation

    Validation and expression analyses of GRB2 and phosphoproteins in breast cancer cells that are associated by interactions on the phosphopeptide chip. 5a. Immunoprecipitation and western analysis of endogenous protein complexes bound to phosphoproteins PTPN11pY584 and SHCpY527 from breast normal and cancer cells (MCF10A, MCF7 and MDA-MB231). The top and middle panel shows immunoprecipitated phosphospecific proteins PTPRA pY798 and PTPN11 pY584 while the bottom panel shows the presence of GRB2 (blotted with monoclonal anti- mouse GRB2 antibody) in immunoprecipitates of PTPN11 pY584 and SHC pY427. 5b. Immunoprecipitation and western analysis of protein complexes bound to specific phosphoproteins in breast cancer cells (MCF10A and T47D) used in the study. Analysis of immunoprecipitates of phospho-specific antibody (VEGFR1 pY1213) and EGFR pY1092): Top panel shows the presence of phospho-VEGFR1 (pY1213) proteins in MCF10A (lanes 3 and 4) and T47D (lanes 6 and 7). The intensity of bands probably reflects the expression levels of VEGFR1. The bottom panel shows the presence of GRB2 in VEGFR1 immunoprecipitates but not in EGFR1 immunoprecipitates. In case of EGFR1 (pY1092) phosphoprotein the interaction of GRB2 may be indirect involving an unknown sandwich protein. 5c. Analysis of level of expression of phosphoproteins used in the chip assay and cell based protein interaction studies. The expression levels were almost similar for PTPN11 pY584 and SHC pY427, but the difference was evident in case of EGFR pY1092, PTPRA pY798 and VEGFR pY1213 which is very minimal in MCF7 and T47D compared to MCF10A and MDA-MB231 but the GRB2 binding was much higher in chip assays with MCF7 and T47D. This indicates that the upregulation we see in chip assays might involve an increase in stoichiometry of tyrosine phosphorylation in tumor cells compared to the normal and metastatic cells. 5d. The expression level of GRB2 indicated that the endogenous GRB2 is low abundant but the level of expression is upregulated in one of tumor cells (MCF7) followed by T47D which indicates that the upregulation of GRB2 binding in chip assays in tumor cells could be because of increased expression of GRB2 in these cells.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Validation and expression analyses of GRB2 and phosphoproteins in breast cancer cells that are associated by interactions on the phosphopeptide chip. 5a. Immunoprecipitation and western analysis of endogenous protein complexes bound to phosphoproteins PTPN11pY584 and SHCpY527 from breast normal and cancer cells (MCF10A, MCF7 and MDA-MB231). The top and middle panel shows immunoprecipitated phosphospecific proteins PTPRA pY798 and PTPN11 pY584 while the bottom panel shows the presence of GRB2 (blotted with monoclonal anti- mouse GRB2 antibody) in immunoprecipitates of PTPN11 pY584 and SHC pY427. 5b. Immunoprecipitation and western analysis of protein complexes bound to specific phosphoproteins in breast cancer cells (MCF10A and T47D) used in the study. Analysis of immunoprecipitates of phospho-specific antibody (VEGFR1 pY1213) and EGFR pY1092): Top panel shows the presence of phospho-VEGFR1 (pY1213) proteins in MCF10A (lanes 3 and 4) and T47D (lanes 6 and 7). The intensity of bands probably reflects the expression levels of VEGFR1. The bottom panel shows the presence of GRB2 in VEGFR1 immunoprecipitates but not in EGFR1 immunoprecipitates. In case of EGFR1 (pY1092) phosphoprotein the interaction of GRB2 may be indirect involving an unknown sandwich protein. 5c. Analysis of level of expression of phosphoproteins used in the chip assay and cell based protein interaction studies. The expression levels were almost similar for PTPN11 pY584 and SHC pY427, but the difference was evident in case of EGFR pY1092, PTPRA pY798 and VEGFR pY1213 which is very minimal in MCF7 and T47D compared to MCF10A and MDA-MB231 but the GRB2 binding was much higher in chip assays with MCF7 and T47D. This indicates that the upregulation we see in chip assays might involve an increase in stoichiometry of tyrosine phosphorylation in tumor cells compared to the normal and metastatic cells. 5d. The expression level of GRB2 indicated that the endogenous GRB2 is low abundant but the level of expression is upregulated in one of tumor cells (MCF7) followed by T47D which indicates that the upregulation of GRB2 binding in chip assays in tumor cells could be because of increased expression of GRB2 in these cells.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Binding Assay

    Mapping the strength of association of each tyrosine phosphomotif relevant to RTK pathways with GRB2 from normal breast cells and tumor cells. Comparative map of active pY motifs on various RTK signaling proteins in breast normal and cancer cells: MCF10A, MCF7, T47D and MDA-MB231. Each bar graph shows relative strengths of each of the tyrosine phospho-motif present in selected RTK pathway proteins interacting with GRB2 as measured by the signal intensity of GRB2 binding to Phosphopeptides (PPEPs) on microarray. The Y axis indicates all the phospho-motifs (source: Phosphosite) on the protein from all four cell lines compared with a control peptide (e.g.EGFR pY988 Vs EGFR Y988A). The X axis indicate intensity of GRB2 binding to each phosphopeptide probes (pY and A peptides). Each value is an average of three independent observations.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Mapping the strength of association of each tyrosine phosphomotif relevant to RTK pathways with GRB2 from normal breast cells and tumor cells. Comparative map of active pY motifs on various RTK signaling proteins in breast normal and cancer cells: MCF10A, MCF7, T47D and MDA-MB231. Each bar graph shows relative strengths of each of the tyrosine phospho-motif present in selected RTK pathway proteins interacting with GRB2 as measured by the signal intensity of GRB2 binding to Phosphopeptides (PPEPs) on microarray. The Y axis indicates all the phospho-motifs (source: Phosphosite) on the protein from all four cell lines compared with a control peptide (e.g.EGFR pY988 Vs EGFR Y988A). The X axis indicate intensity of GRB2 binding to each phosphopeptide probes (pY and A peptides). Each value is an average of three independent observations.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Multiple Displacement Amplification, Binding Assay, Microarray

    Phospho-PepArray analysis of signaling interactome from breast cancer cells. (a) Steps in phospho-motif binding assay of endogenous cellular protein complexes in cells: (a) cells are cultured on plates. (b) Total protein is isolated from cultured cells after cell lysis and cell lysate is applied to the phosphopeptides synthesized on (c) PepArray chip through microfluidics by circulation at 4°C overnight. (d) Antibody based detection is used to identify the protein of interest in these complexes. A general detection method is to stain the binding surface using anti-GRB2 antibody and a fluorescence dye conjugated secondary antibody such as Alexa. Based on in vivo substrate affinity of a specific phosphoprotein motif with binding domains on other cellular proteins, in vivo protein complexes, from the pool of non-denatured total proteins, are bound to respective phospho-peptides (pY) on the chip. 3(b) Illustration of the possible peptide probe interaction with endogenous protein complexes from cells due to inter-protein interactions: Endogenous protein complexes containing SH2 domain in cell total proteome can bind to directly or indirectly to phosphopeptides (PPEPs) on the chip. For example GRB2, an SH2 domain containing protein can either directly bind to a phosphopeptide probe through the SH2 domain or can indirectly bind to the PPEP through interacting with the pY sites of a sandwich protein (Protein X) which is bound to PPEP directly through its SH2 domain. Another way of indirect interaction of GBR2 is through the SH3 domain (bind to poly-proline-rich regions) that might interact with ploy proline rich region of the sandwich protein bound to the pY peptide probe. The presence of GRB2 either by direct or indirect interaction with a pY protein trapped on the respective phosphopeptide probe on the chip results in an interaction signal detected by florescent conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Phospho-PepArray analysis of signaling interactome from breast cancer cells. (a) Steps in phospho-motif binding assay of endogenous cellular protein complexes in cells: (a) cells are cultured on plates. (b) Total protein is isolated from cultured cells after cell lysis and cell lysate is applied to the phosphopeptides synthesized on (c) PepArray chip through microfluidics by circulation at 4°C overnight. (d) Antibody based detection is used to identify the protein of interest in these complexes. A general detection method is to stain the binding surface using anti-GRB2 antibody and a fluorescence dye conjugated secondary antibody such as Alexa. Based on in vivo substrate affinity of a specific phosphoprotein motif with binding domains on other cellular proteins, in vivo protein complexes, from the pool of non-denatured total proteins, are bound to respective phospho-peptides (pY) on the chip. 3(b) Illustration of the possible peptide probe interaction with endogenous protein complexes from cells due to inter-protein interactions: Endogenous protein complexes containing SH2 domain in cell total proteome can bind to directly or indirectly to phosphopeptides (PPEPs) on the chip. For example GRB2, an SH2 domain containing protein can either directly bind to a phosphopeptide probe through the SH2 domain or can indirectly bind to the PPEP through interacting with the pY sites of a sandwich protein (Protein X) which is bound to PPEP directly through its SH2 domain. Another way of indirect interaction of GBR2 is through the SH3 domain (bind to poly-proline-rich regions) that might interact with ploy proline rich region of the sandwich protein bound to the pY peptide probe. The presence of GRB2 either by direct or indirect interaction with a pY protein trapped on the respective phosphopeptide probe on the chip results in an interaction signal detected by florescent conjugated secondary antibody.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Binding Assay, Cell Culture, Isolation, Lysis, Synthesized, Chromatin Immunoprecipitation, Staining, Fluorescence, In Vivo

    Pathway interactome of upregulated GRB2-pY proteome interactome in breast tumor cells (MCF7 and T47D). The GRB2-pY proteome interactions imply cross-talk between various signaling pathways that are functional to initiate various cellular responses. The GBR2 interactome revealed connections of RTK pathways with both classical GRB2-SOS-RAS cascade with PI3K-IRS1 cascade leading to AKT and ERK activation. The crosstalk between growth factor and cytokine signaling pathways as revealed by major receptors and adaptors and other downstream proteins. Upregulation of negative regulation of immune signaling pathways in tumor cells indicate the preparedness of tumor cells to fight and survive attack from host immune signaling mechanisms. Novel interactions are marked by red arrows.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Pathway interactome of upregulated GRB2-pY proteome interactome in breast tumor cells (MCF7 and T47D). The GRB2-pY proteome interactions imply cross-talk between various signaling pathways that are functional to initiate various cellular responses. The GBR2 interactome revealed connections of RTK pathways with both classical GRB2-SOS-RAS cascade with PI3K-IRS1 cascade leading to AKT and ERK activation. The crosstalk between growth factor and cytokine signaling pathways as revealed by major receptors and adaptors and other downstream proteins. Upregulation of negative regulation of immune signaling pathways in tumor cells indicate the preparedness of tumor cells to fight and survive attack from host immune signaling mechanisms. Novel interactions are marked by red arrows.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Functional Assay, Activation Assay

    Highlights of the results of total cellular protein assay on pY peptide microarray. ( a ) Phosphopeptide microarray images showing spots of GRB2 association with specific phosphopeptide probes that represent phospho-motifs that are upregulated in ER positive breast tumor cells (MCF7 and T47D) compared to normal (MCF10A) or metastatic (MDA-MB231) cells that are ER negative. ( b ) Heatmap comparing high confidence GRB2 interactions from normal breast epithelial (MCF10A), breast tumor (MCF7, T47D) and metastatic (MDA-MB231) cell lines. The binding signals were normalized into Z values. The PPEPs with Z values ranked from 1.0–3.0 were selected to plot the heat map. The heat map clearly indicates upregulation of GRB2 interactome in tumor cells compared to normal or metastatic cells.

    Journal: PLoS ONE

    Article Title: A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks

    doi: 10.1371/journal.pone.0067634

    Figure Lengend Snippet: Highlights of the results of total cellular protein assay on pY peptide microarray. ( a ) Phosphopeptide microarray images showing spots of GRB2 association with specific phosphopeptide probes that represent phospho-motifs that are upregulated in ER positive breast tumor cells (MCF7 and T47D) compared to normal (MCF10A) or metastatic (MDA-MB231) cells that are ER negative. ( b ) Heatmap comparing high confidence GRB2 interactions from normal breast epithelial (MCF10A), breast tumor (MCF7, T47D) and metastatic (MDA-MB231) cell lines. The binding signals were normalized into Z values. The PPEPs with Z values ranked from 1.0–3.0 were selected to plot the heat map. The heat map clearly indicates upregulation of GRB2 interactome in tumor cells compared to normal or metastatic cells.

    Article Snippet: For Cell lysate binding assays Rabbit-Anti-GRB2 and Mouse-Anti- SRC (Cell Signaling Technology, Beverley, MA) were used primary antibodies at a dilution of 1∶1000 and 1∶500 respectively.

    Techniques: Peptide Microarray, Microarray, Multiple Displacement Amplification, Binding Assay

    Panx1 and Panx3 are expressed in the skeletal muscle tissue. A , expression of Panx1, Panx2, and Panx3 proteins in adult human, mouse, and rat skeletal muscle whole tissue lysates was analyzed by Western blotting. Various molecular weight species of Panx1 and Panx3 were expressed, whereas Panx2 was absent or below detectable levels. HEK293T cells transfected with Panx1, Panx2, or Panx3 were used as positive controls. Desmin is a muscle-specific protein. B , human skeletal muscle tissue in skin samples was labeled for Panx1 (labeled in red ) and Panx3 (labeled in red ). Representative images are shown. Panx1 was detected as a punctate stain, whereas Panx3 was observed as diffuse labeling. Higher magnification micrographs of Panx3 labeling show a striated pattern. Peptide competition revealed loss of specific staining. Blue = nuclei; bars = 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

    doi: 10.1074/jbc.M114.572131

    Figure Lengend Snippet: Panx1 and Panx3 are expressed in the skeletal muscle tissue. A , expression of Panx1, Panx2, and Panx3 proteins in adult human, mouse, and rat skeletal muscle whole tissue lysates was analyzed by Western blotting. Various molecular weight species of Panx1 and Panx3 were expressed, whereas Panx2 was absent or below detectable levels. HEK293T cells transfected with Panx1, Panx2, or Panx3 were used as positive controls. Desmin is a muscle-specific protein. B , human skeletal muscle tissue in skin samples was labeled for Panx1 (labeled in red ) and Panx3 (labeled in red ). Representative images are shown. Panx1 was detected as a punctate stain, whereas Panx3 was observed as diffuse labeling. Higher magnification micrographs of Panx3 labeling show a striated pattern. Peptide competition revealed loss of specific staining. Blue = nuclei; bars = 50 μm.

    Article Snippet: Normal fetal and adult human skeletal muscle whole lysates were obtained from Novus Biologicals (Oakville, ON, Canada).

    Techniques: Expressing, Western Blot, Molecular Weight, Transfection, Labeling, Staining

    Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation. A , Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified ( B and C ). For both Panx1 and Panx3, the higher M r species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower M r forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM ( D ) and SkMC ( G ) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation ( E and H ), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation ( F and I ). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

    doi: 10.1074/jbc.M114.572131

    Figure Lengend Snippet: Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation. A , Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified ( B and C ). For both Panx1 and Panx3, the higher M r species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower M r forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM ( D ) and SkMC ( G ) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation ( E and H ), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation ( F and I ). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.

    Article Snippet: Normal fetal and adult human skeletal muscle whole lysates were obtained from Novus Biologicals (Oakville, ON, Canada).

    Techniques: Expressing

    Panx1 and Panx3 levels are modulated during skeletal muscle myoblast differentiation and regulate this process as well as myoblast proliferation. Proliferative undifferentiated skeletal muscle myoblasts express high levels of the ∼70-kDa immunoreactive species of Panx3, whereas its lower M r species or Panx1 were very low. Its levels were drastically diminished during differentiation, suggesting that the ∼70-kDa immunoreactive species of Panx3 may play a role in keeping undifferentiated skeletal muscle myoblasts in a proliferative state. On the other hand, the levels of the low M r form of Panx3 are very low in skeletal muscle myoblasts in culture, slightly detected in fetal skeletal muscle tissue, and further increased in the adult. Its expression promotes human skeletal muscle myoblast differentiation and inhibits their proliferation. Altogether, our data suggest that the lower M r species of Panx3 play a role in maintaining differentiated myoblasts in a differentiated and non-proliferative state. As for Panx1, its protein levels were drastically augmented during skeletal muscle myoblast differentiation and promote this process independently of cell proliferation.

    Journal: The Journal of Biological Chemistry

    Article Title: Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

    doi: 10.1074/jbc.M114.572131

    Figure Lengend Snippet: Panx1 and Panx3 levels are modulated during skeletal muscle myoblast differentiation and regulate this process as well as myoblast proliferation. Proliferative undifferentiated skeletal muscle myoblasts express high levels of the ∼70-kDa immunoreactive species of Panx3, whereas its lower M r species or Panx1 were very low. Its levels were drastically diminished during differentiation, suggesting that the ∼70-kDa immunoreactive species of Panx3 may play a role in keeping undifferentiated skeletal muscle myoblasts in a proliferative state. On the other hand, the levels of the low M r form of Panx3 are very low in skeletal muscle myoblasts in culture, slightly detected in fetal skeletal muscle tissue, and further increased in the adult. Its expression promotes human skeletal muscle myoblast differentiation and inhibits their proliferation. Altogether, our data suggest that the lower M r species of Panx3 play a role in maintaining differentiated myoblasts in a differentiated and non-proliferative state. As for Panx1, its protein levels were drastically augmented during skeletal muscle myoblast differentiation and promote this process independently of cell proliferation.

    Article Snippet: Normal fetal and adult human skeletal muscle whole lysates were obtained from Novus Biologicals (Oakville, ON, Canada).

    Techniques: Expressing

    Treatment with N -glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3. A , deglycosylation using a mix of enzymes ( N -glycosidase F, O -glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β- N -acetylglucosaminidase) caused a shift in electrophoretic mobility ( arrow ) of the higher M r form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B , incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility ( arrow ) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower M r form of Panx3 transfected in HEK293T cells. C , treatment with N -glycosidase F ( PNGase F ) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O -glycanase, β(1–4)-galactosidase, or β- N -acetylglucosaminidase.

    Journal: The Journal of Biological Chemistry

    Article Title: Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

    doi: 10.1074/jbc.M114.572131

    Figure Lengend Snippet: Treatment with N -glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3. A , deglycosylation using a mix of enzymes ( N -glycosidase F, O -glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β- N -acetylglucosaminidase) caused a shift in electrophoretic mobility ( arrow ) of the higher M r form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B , incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility ( arrow ) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower M r form of Panx3 transfected in HEK293T cells. C , treatment with N -glycosidase F ( PNGase F ) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O -glycanase, β(1–4)-galactosidase, or β- N -acetylglucosaminidase.

    Article Snippet: Normal fetal and adult human skeletal muscle whole lysates were obtained from Novus Biologicals (Oakville, ON, Canada).

    Techniques: Transfection, Incubation

    Inhibition of both HIF1α and HIF-2α diminishes Bcl-2 levels without affecting BAX levels. Cell lysates collected from cells treated with 0.5 µM, 5 µM, and 50 µM of HIF-1α/HIF-2α translation inhibitors were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX, and a significant decrease in the levels of Bcl-2 coupled with the loss of VEGF ( Figure 4L ) was observed with the double translation inhibitor ( A ). B and C represent the corresponding densitometry analysis.

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: Inhibition of both HIF1α and HIF-2α diminishes Bcl-2 levels without affecting BAX levels. Cell lysates collected from cells treated with 0.5 µM, 5 µM, and 50 µM of HIF-1α/HIF-2α translation inhibitors were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX, and a significant decrease in the levels of Bcl-2 coupled with the loss of VEGF ( Figure 4L ) was observed with the double translation inhibitor ( A ). B and C represent the corresponding densitometry analysis.

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Western Blot

    Inhibition of HIF-2α does not influence BAX or Bcl-2 levels. Cell lysates collected from cells treated with 0.5 µM, 5 µM, and 50 µM of HIF-2α translation inhibitor were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX and Bcl-2 ( A ). B and C represent the corresponding densitometry analysis.

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: Inhibition of HIF-2α does not influence BAX or Bcl-2 levels. Cell lysates collected from cells treated with 0.5 µM, 5 µM, and 50 µM of HIF-2α translation inhibitor were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX and Bcl-2 ( A ). B and C represent the corresponding densitometry analysis.

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Western Blot

    HIF-1α inhibition does not affect VEGF expression. A : western blot analysis of HIF-1α expression in HLE-B3 cells treated with topotecan. Cell lysates were collected from cells treated with 500 nM topotecan in 0.01% DMSO after 8 h of hypoxic incubation. Control cells were mock treated with 0.01% DMSO and maintained in hypoxia as the topotecan-treated cells. Twenty μg protein/lane of cell lysates were analyzed with western blot analysis, and lane loading was normalized using a 1:1,000 dilution of rabbit anti-pan-actin antibody. Topotecan inhibited the expression of HIF-1α (1:1,000 dilution of rabbit anti- HIF-1α antibody) while a compensatory increase in HIF-2α was noted (1:1,000 dilution of rabbit anti- HIF-2α antibody). B, C : Densitometry analysis of HIF-1α and HIF-2α, respectively. D : Effect of HIF-1α inhibition on VEGF synthesis in hypoxia. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 500 nM topotecan or 0.01% DMSO for 8 h of hypoxic exposure. Cell-free supernatants were collected in triplicate at the end of hypoxic incubation and analyzed for VEGF levels with ELISA. There was no significant difference in the VEGF levels between the topotecan-treated cells and control cells. (A Student t test was performed to compare the VEGF levels between control and treated sample, and the p value was greater than 0.05.) E : HIF-2α protein from HLE-B3 control cells compared with a standard lysate prepared from human embryonic kidney cells (HEK) 293 (Novus Biologicals, Litton, CO). The HIF-2α found in the standard lysate migrated at about 97 kDa. The HIF-2 α from HLE-B3 cells was about 80 kDa.

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: HIF-1α inhibition does not affect VEGF expression. A : western blot analysis of HIF-1α expression in HLE-B3 cells treated with topotecan. Cell lysates were collected from cells treated with 500 nM topotecan in 0.01% DMSO after 8 h of hypoxic incubation. Control cells were mock treated with 0.01% DMSO and maintained in hypoxia as the topotecan-treated cells. Twenty μg protein/lane of cell lysates were analyzed with western blot analysis, and lane loading was normalized using a 1:1,000 dilution of rabbit anti-pan-actin antibody. Topotecan inhibited the expression of HIF-1α (1:1,000 dilution of rabbit anti- HIF-1α antibody) while a compensatory increase in HIF-2α was noted (1:1,000 dilution of rabbit anti- HIF-2α antibody). B, C : Densitometry analysis of HIF-1α and HIF-2α, respectively. D : Effect of HIF-1α inhibition on VEGF synthesis in hypoxia. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 500 nM topotecan or 0.01% DMSO for 8 h of hypoxic exposure. Cell-free supernatants were collected in triplicate at the end of hypoxic incubation and analyzed for VEGF levels with ELISA. There was no significant difference in the VEGF levels between the topotecan-treated cells and control cells. (A Student t test was performed to compare the VEGF levels between control and treated sample, and the p value was greater than 0.05.) E : HIF-2α protein from HLE-B3 control cells compared with a standard lysate prepared from human embryonic kidney cells (HEK) 293 (Novus Biologicals, Litton, CO). The HIF-2α found in the standard lysate migrated at about 97 kDa. The HIF-2 α from HLE-B3 cells was about 80 kDa.

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Expressing, Western Blot, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Inhibition of both HIF-1α and HIF-2α elicits mitochondrial membrane depolarization. Cells were treated with 50 μM of HIF-1α/HIF-2α translation inhibitor in 0.05% DMSO for 3 h in hypoxia. Control cells were treated with 0.05% DMSO. After the hypoxic exposure, the media was replaced with fresh oxygenated serum-free media containing 5 µg/ml JC-1 for 30 min in atmospheric oxygen. The media were removed, and fresh serum-free media were added to the cells. Control cells incubated with DMSO only were treated in a similar manner. We used serial confocal imaging to monitor mitochondrial membrane depolarization in HLE-B3 cells after treatment with the HIF-1α/HIF-2α double translation inhibitor. Sequential images of a random field of cells were taken every 150 s throughout the 60 min duration (Bar=20 µm). Confocal images of the HIF-1α/HIF-2α double translation inhibitor-treated cells indicated that there was a marked increase in green fluorescence intensity (indicative of depolarization) at both 30 and 60 min ( B ) compared with the control cells ( A ). C : HLE-B3 cells treated with the HIF-1α/HIF-2α double translation inhibitor exhibited a significantly increased green/red ratio compared with control, untreated cells.

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: Inhibition of both HIF-1α and HIF-2α elicits mitochondrial membrane depolarization. Cells were treated with 50 μM of HIF-1α/HIF-2α translation inhibitor in 0.05% DMSO for 3 h in hypoxia. Control cells were treated with 0.05% DMSO. After the hypoxic exposure, the media was replaced with fresh oxygenated serum-free media containing 5 µg/ml JC-1 for 30 min in atmospheric oxygen. The media were removed, and fresh serum-free media were added to the cells. Control cells incubated with DMSO only were treated in a similar manner. We used serial confocal imaging to monitor mitochondrial membrane depolarization in HLE-B3 cells after treatment with the HIF-1α/HIF-2α double translation inhibitor. Sequential images of a random field of cells were taken every 150 s throughout the 60 min duration (Bar=20 µm). Confocal images of the HIF-1α/HIF-2α double translation inhibitor-treated cells indicated that there was a marked increase in green fluorescence intensity (indicative of depolarization) at both 30 and 60 min ( B ) compared with the control cells ( A ). C : HLE-B3 cells treated with the HIF-1α/HIF-2α double translation inhibitor exhibited a significantly increased green/red ratio compared with control, untreated cells.

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Incubation, Imaging, Fluorescence

    Inhibition of both HIF-1α and HIF-2α elicits the loss of VEGF expression. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 0.5 µm, 5 µm, and 50 µm HIF-1α inhibitor, HIF-2α inhibitor, and HIF-1α/HIF-2α double translation inhibitor for 3 or 8 h in hypoxia. The effect of the inhibitors on HIF-1α and HIF-2α protein expression was analyzed using western blot analysis. Cell lysates (20 ug protein/lane) were identified using either anti-rabbit HIF-1α or HIF-2α at 1:1000 dilutions and lane loading was normalized using a 1:1000 dilution of rabbit anti- pan-actin antibody. Effect of HIF-1α and/or HIF-2α inhibition on VEGF levels in hypoxia. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 0.5 µm, 5 µm and 50 µm HIF-1α inhibitor, HIF-2α inhibitor and HIF-1α/HIF-2α double translation inhibitor for 3 or 8 h in hypoxia. Cell free supernatants collected in triplicate were analyzed for VEGF levels by ELISA. The HIF-1α translation inhibitor at all concentrations inhibited HIF-1α without affecting the HIF-2α protein synthesis ( A ). Figure ( B ) and ( C ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was no significant difference in the VEGF levels between the control cells and cells treated with HIF-1α inhibitor ( D ). One-way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-1α inhibitor and the p value was > 0.05. The HIF-2α translation inhibitor at all concentrations inhibited HIF-2α without affecting the HIF-1α protein synthesis ( E ). Figure ( F ) and ( G ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was no significant difference in the VEGF levels between the control cells and cells treated with HIF-2α inhibitor ( H ). One -way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-2α inhibitor and the p value was > 0.05.The HIF-1α/HIF-2α double translation inhibitor at all concentrations inhibited HIF-2α and HIF-1α protein synthesis ( I ). Figure ( J ) and ( K ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was significant difference in the VEGF levels between the control cells and cells treated with HIF-1α/ HIF-2α double translation inhibitor at 3 h and 8 h of hypoxia. L : One-way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-1α/ HIF-2α double translation inhibitor and the p value was

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: Inhibition of both HIF-1α and HIF-2α elicits the loss of VEGF expression. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 0.5 µm, 5 µm, and 50 µm HIF-1α inhibitor, HIF-2α inhibitor, and HIF-1α/HIF-2α double translation inhibitor for 3 or 8 h in hypoxia. The effect of the inhibitors on HIF-1α and HIF-2α protein expression was analyzed using western blot analysis. Cell lysates (20 ug protein/lane) were identified using either anti-rabbit HIF-1α or HIF-2α at 1:1000 dilutions and lane loading was normalized using a 1:1000 dilution of rabbit anti- pan-actin antibody. Effect of HIF-1α and/or HIF-2α inhibition on VEGF levels in hypoxia. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 0.5 µm, 5 µm and 50 µm HIF-1α inhibitor, HIF-2α inhibitor and HIF-1α/HIF-2α double translation inhibitor for 3 or 8 h in hypoxia. Cell free supernatants collected in triplicate were analyzed for VEGF levels by ELISA. The HIF-1α translation inhibitor at all concentrations inhibited HIF-1α without affecting the HIF-2α protein synthesis ( A ). Figure ( B ) and ( C ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was no significant difference in the VEGF levels between the control cells and cells treated with HIF-1α inhibitor ( D ). One-way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-1α inhibitor and the p value was > 0.05. The HIF-2α translation inhibitor at all concentrations inhibited HIF-2α without affecting the HIF-1α protein synthesis ( E ). Figure ( F ) and ( G ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was no significant difference in the VEGF levels between the control cells and cells treated with HIF-2α inhibitor ( H ). One -way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-2α inhibitor and the p value was > 0.05.The HIF-1α/HIF-2α double translation inhibitor at all concentrations inhibited HIF-2α and HIF-1α protein synthesis ( I ). Figure ( J ) and ( K ) represent the densitometry analysis for HIF-1α and HIF-2α protein expression. There was significant difference in the VEGF levels between the control cells and cells treated with HIF-1α/ HIF-2α double translation inhibitor at 3 h and 8 h of hypoxia. L : One-way ANOVA analysis was performed to compare the VEGF levels between the control and the three concentrations of HIF-1α/ HIF-2α double translation inhibitor and the p value was

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Expressing, Cell Culture, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

    Inhibition of pERK or pAkt does not affect VEGF expression. Western blot analysis of ERK1/2 and Akt phosphorylation inhibition on HIF-1α, HIF-2α, or downstream VEGF expression in hypoxia. HLE-B3 cells were incubated with 25 µM of LY294002 or 10 µM UO126 in 0.05% DMSO for 8 h under hypoxia. Control cells were incubated in serum-free media with 0.05% DMSO. Cell lysates were collected for western blot analysis, and cell-free supernatants were collected for ELISA. LY294002 blocked Akt phosphorylation without interrupting pERK, while UO126 prevented ERK phosphorylation without interfering with pAkt ( A ). Inhibition of Akt phosphorylation suppressed HIF-1α expression, but HIF-2α levels remained unchanged. Inhibition of ERK1/2 phosphorylation did not repress HIF-1α and HIF-2α expression ( B ). C and D represent the corresponding densitometry analysis. Neither phosphorylation inhibitor diminished VEGF synthesis relative to control cells ( E ). One-way ANOVA analysis was performed to compare the VEGF levels between the control and the LY294002- or UO126-treated samples, and the p value was > 0.05.

    Journal: Molecular Vision

    Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1? and -2? and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

    doi:

    Figure Lengend Snippet: Inhibition of pERK or pAkt does not affect VEGF expression. Western blot analysis of ERK1/2 and Akt phosphorylation inhibition on HIF-1α, HIF-2α, or downstream VEGF expression in hypoxia. HLE-B3 cells were incubated with 25 µM of LY294002 or 10 µM UO126 in 0.05% DMSO for 8 h under hypoxia. Control cells were incubated in serum-free media with 0.05% DMSO. Cell lysates were collected for western blot analysis, and cell-free supernatants were collected for ELISA. LY294002 blocked Akt phosphorylation without interrupting pERK, while UO126 prevented ERK phosphorylation without interfering with pAkt ( A ). Inhibition of Akt phosphorylation suppressed HIF-1α expression, but HIF-2α levels remained unchanged. Inhibition of ERK1/2 phosphorylation did not repress HIF-1α and HIF-2α expression ( B ). C and D represent the corresponding densitometry analysis. Neither phosphorylation inhibitor diminished VEGF synthesis relative to control cells ( E ). One-way ANOVA analysis was performed to compare the VEGF levels between the control and the LY294002- or UO126-treated samples, and the p value was > 0.05.

    Article Snippet: To further confirm the identity of HIF-2α in HLE-B3 cells, a HIF-2α standard lysate (Novus Biologicals) was compared with lysate from HLE-B3 cells.

    Techniques: Inhibition, Expressing, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

    Outputs of proteomic analysis for CA-altered proteins. (A) Venn diagram showing numerical distribution of proteins identified in different RAW264.7 cell lysates by the nanoLC-LTQ-Orbitrap MS/MS approach. Cells were treated with vehicle (control group), LPS (1 μg/ml, LPS group), and LPS with 20 μM of CA (LPS+CA group) for 6 h, respectively. (B–D) Proteins significantly up-regulated and down-regulated by CA were classified into different cellular components (B) , molecular functions (C) , and BPs (D) .

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: Outputs of proteomic analysis for CA-altered proteins. (A) Venn diagram showing numerical distribution of proteins identified in different RAW264.7 cell lysates by the nanoLC-LTQ-Orbitrap MS/MS approach. Cells were treated with vehicle (control group), LPS (1 μg/ml, LPS group), and LPS with 20 μM of CA (LPS+CA group) for 6 h, respectively. (B–D) Proteins significantly up-regulated and down-regulated by CA were classified into different cellular components (B) , molecular functions (C) , and BPs (D) .

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Mass Spectrometry

    Carnosic acid down-regulated the levels of pro-inflammation gene expression in LPS-stimulated RAW264.7 cells. (A–D) Cells were treated with LPS (1 μg/ml) for 6 h with or without CA (5, 10, and 20 μM). The relative mRNA expressions of Nos2 (A) , Tnfα (B) , Cox2 (C) , and Mcp1 (D) were detected by real-time PCR analysis, respectively. Data are expressed as mean ± SEM ( n = 3). ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: Carnosic acid down-regulated the levels of pro-inflammation gene expression in LPS-stimulated RAW264.7 cells. (A–D) Cells were treated with LPS (1 μg/ml) for 6 h with or without CA (5, 10, and 20 μM). The relative mRNA expressions of Nos2 (A) , Tnfα (B) , Cox2 (C) , and Mcp1 (D) were detected by real-time PCR analysis, respectively. Data are expressed as mean ± SEM ( n = 3). ∗ P

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    CA restrained the activation of ERK, JNK, and p38 MAPKs in LPS-challenged RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylations of ERK, JNK, and p38 protein were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of ERK (B) , JNK (C) , and p38 MAPK (D) was performed by normalizing to the control group. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: CA restrained the activation of ERK, JNK, and p38 MAPKs in LPS-challenged RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylations of ERK, JNK, and p38 protein were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of ERK (B) , JNK (C) , and p38 MAPK (D) was performed by normalizing to the control group. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Activation Assay, Western Blot

    Bioinformatics analysis of the signaling networks negatively regulated by CA. (A,B) Pathway enrichment analysis for the differentially expressed proteins predicted the significantly canonical pathways targeted by CA in LPS-stimulated RAW264.7 cells. The gray columns plotting on the left Y -axis depict the number of identified proteins found in each pathway. The olive yellow columns plotting on the right Y -axis depict the percentage of identified proteins over the total proteins in that pathway. (C) BP enrichment analysis for differentially expressed proteins in CA-treated RAW264.7 cells.

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: Bioinformatics analysis of the signaling networks negatively regulated by CA. (A,B) Pathway enrichment analysis for the differentially expressed proteins predicted the significantly canonical pathways targeted by CA in LPS-stimulated RAW264.7 cells. The gray columns plotting on the left Y -axis depict the number of identified proteins found in each pathway. The olive yellow columns plotting on the right Y -axis depict the percentage of identified proteins over the total proteins in that pathway. (C) BP enrichment analysis for differentially expressed proteins in CA-treated RAW264.7 cells.

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques:

    CA suppressed LPS-induced IKKβ/IκB-α/NF-κB signaling activation in RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylation and total expressions of IKKβ, IκBα, and NF-κB p65 were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of IKKβ (B) , IκB-α (C) , and NF-κB p65 (D) was performed by normalizing to the control group. (E) The nuclear translocation of NF-κB p65 was detected by immunofluorescence assay. Representative images were displayed with NF-κB p65 (red) and nucleus (blue). Typical apoptotic neurons were labeled with white arrows. Scale bar = 40 μm. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: CA suppressed LPS-induced IKKβ/IκB-α/NF-κB signaling activation in RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylation and total expressions of IKKβ, IκBα, and NF-κB p65 were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of IKKβ (B) , IκB-α (C) , and NF-κB p65 (D) was performed by normalizing to the control group. (E) The nuclear translocation of NF-κB p65 was detected by immunofluorescence assay. Representative images were displayed with NF-κB p65 (red) and nucleus (blue). Typical apoptotic neurons were labeled with white arrows. Scale bar = 40 μm. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Activation Assay, Western Blot, Translocation Assay, Immunofluorescence, Labeling

    Carnosic acid down-regulated the levels of pro-inflammation mediators in LPS-induced RAW264.7 cells. (A) Chemical structure of carnosic acid (CA). (B,C) RAW264.7 cells were treated with various concentration of CA (2.5, 5, 10, and 20 μM) in the absence or presence of LPS (1 μg/ml) for 24 h. Then the cell viability (B) and nitric oxide production (C) were determined by MTT and Griess methods, respectively. (D) RAW274.7 cells were treated with CA and LPS as in (B,C) for 4 h, then the TNF-α level was detected by ELISA. (E) RAW264.7 cells were treated with 1 μg/ml of LPS containing CA (2.5, 5, 10, and 20 μM) or not for 24 h. The protein expression of COX2 was detected by western blot assay. Data are expressed as mean ± SEM from three individual experiments. ∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: Carnosic acid down-regulated the levels of pro-inflammation mediators in LPS-induced RAW264.7 cells. (A) Chemical structure of carnosic acid (CA). (B,C) RAW264.7 cells were treated with various concentration of CA (2.5, 5, 10, and 20 μM) in the absence or presence of LPS (1 μg/ml) for 24 h. Then the cell viability (B) and nitric oxide production (C) were determined by MTT and Griess methods, respectively. (D) RAW274.7 cells were treated with CA and LPS as in (B,C) for 4 h, then the TNF-α level was detected by ELISA. (E) RAW264.7 cells were treated with 1 μg/ml of LPS containing CA (2.5, 5, 10, and 20 μM) or not for 24 h. The protein expression of COX2 was detected by western blot assay. Data are expressed as mean ± SEM from three individual experiments. ∗∗ P

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Concentration Assay, MTT Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

    CA prevented FoxO1 and FoxO3 proteins translocating into the nucleus and promoted their degradation under LPS-challenged situation.chi-tang ho (A,B) RAW264.7 cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 24 h. The total expressions of FoxO1 and FoxO3 were determined by western blot assay. (C) Cells treated as in (A) for 6 h were collected and subjected for real-time PCR experiment to detect the relative mRNA expressions of FoxO1 and FoxO3 . (D,E) The total expressions of FoxO1 and FoxO3 in RAW264.7 cells treated with LPS (1 μg/ml), CA (20 μM), and MG132 (1 μM) for 24 h were determined by western blot assay. (F–I) RAW264.7 cells were treated as in (A) for 1 h, then the nuclear and cytoplasmic expressions of FoxO1 and FoxO3 were detected by western blot assay. Histone H3 and α-tubulin were utilized as internal controls for nuclear and cytoplasmic proteins, respectively. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

    doi: 10.3389/fphar.2018.00370

    Figure Lengend Snippet: CA prevented FoxO1 and FoxO3 proteins translocating into the nucleus and promoted their degradation under LPS-challenged situation.chi-tang ho (A,B) RAW264.7 cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 24 h. The total expressions of FoxO1 and FoxO3 were determined by western blot assay. (C) Cells treated as in (A) for 6 h were collected and subjected for real-time PCR experiment to detect the relative mRNA expressions of FoxO1 and FoxO3 . (D,E) The total expressions of FoxO1 and FoxO3 in RAW264.7 cells treated with LPS (1 μg/ml), CA (20 μM), and MG132 (1 μM) for 24 h were determined by western blot assay. (F–I) RAW264.7 cells were treated as in (A) for 1 h, then the nuclear and cytoplasmic expressions of FoxO1 and FoxO3 were detected by western blot assay. Histone H3 and α-tubulin were utilized as internal controls for nuclear and cytoplasmic proteins, respectively. Data are expressed as mean ± SEM from three individual experiments. ∗ P

    Article Snippet: Protein identification of the RAW264.7 cell lysates against the Thermo Proteome Discoverer database showed a total of 3261, 3351, and 3186 proteins were in the control, LPS, and LPS+CA groups, respectively.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction