Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: c-Jun NH2-terminal Kinase Targeting and Phosphorylation of Heat Shock Factor-1 Suppress Its Transcriptional Activity

doi: 10.1074/jbc.m000958200

Figure Lengend Snippet: FIG. 2. JNK targets HSF-1 to the D domain. A, in vivo interaction of ERK1 with HSF-1. Human lung carcinoma cells H1299 stably expressing HA-tagged ERK1 (HE-10) were transiently trans- fected with full-length pcDNA3-HSF-1 ex- pression vector. After 48 h, cells were ly- sed, and ERK1 was immunoprecipitated using anti-ERK1 antibodies. Immunopre- cipitates were analyzed by PAGE and im- munoblotted using anti-HSF-1 antibody (23). B, diagram of truncated wild type HSF-1 and deletion mutants. Numbers above indicate amino acid residues. Num- bers below indicate start and end points of deleted region in mutant. C, the con- served sequence (underlined) of the D do- main in HSF-1 and other transcription factors that are the target of MAPKs. D and E, binding of JNK1 and ERK1 to truncated wild type His-tagged-HSF-1 (1–450) and truncated His-tagged HSF-1 mutant D01 (deleted between amino acid residues 203 and 224). Anti-JNK1 or anti- ERK1 antibodies (Ab) were bound to pro- tein A-Sepharose beads and incubated with unheated control (C) or heated (45 °C for 30 min) H1299 cell lysates sta- bly expressing JNK1 (D) or ERK1 (E). Purified truncated wild type His-HSF-1 (1–450) or truncated His-HSF-1 mutant D01 were added to the protein A/JNK or ERK antibody mix. The immunoprecipi- tated materials were analyzed by PAGE, followed by immunoblotting using anti- HSF-1 antibody. The positions of the pu- rified HSF-1 (1–450) and IgG bands are indicated. F, requirement of binding to HSF-1 for efficient JNK or ERK phospho- rylation. Wild type HSF-1 or mutant D01 were phosphorylated for 5–60 min at 30 °C with immunoprecipitated activated JNK1 or purified active JNK2, ERK1, or ERK2. Samples were analyzed by PAGE, and gels were exposed to x-ray film. The gels were quantitated using PhosphorIm- ager. All experiments were performed at least three times, and results were con- sistent. G, deletion of the amino acid res- idues 203–224 in HSF-1 protein prevents its nuclear localization. Representative immunofluorescence photographs (mag- nification, 31000) of HeLa cells trans- fected with HSF-1-GFP (wild type), or HSF-1-GFP D01 (deleted between amino acid residues 203 and 224). HeLa cells were transiently transfected with the ex- pression vectors. 48 h after transfection, cell were heated at 45 °C for 30 min and allowed to recover at 37 °C for 4 h, which is the best recovery time after heat shock to observe HSF-1 granules. GFP fluo- resces green under blue light. Wt, wild type.

Article Snippet: For phosphorylation experiments, 1 mg of each substrate was phosphorylated with [g-32P]ATP for 20 min at 30 °C with immunoprecipitated JNK1 (C17, Santa Cruz) or purified active JNK2 (UBI), ERK1 (United Biotechnology, Inc.), or ERK2 (New England BioLabs).

Techniques: In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Sequencing, Binding Assay, Incubation, Control, Purification, Western Blot, Immunofluorescence, Transfection

Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Impaired AJC formation in Albatross knockdown cells. (A) Double staining for Albatross (red) and the undercoat proteins (green) for each AJC component: TJ, ZO-1; AJ, afadin; DS, desmoplakin. Top and bottom columns show projections of x-y planes and z sections, respectively. Albatross knockdown A549 (Albatross KD) cells lack accumulation of these proteins at the cell–cell borders except in regions where residual Albatross is present. (B) Cell–cell adhesive properties evaluated by a cell aggregation assay. In the differential interference contrast images, control cells show cell aggregation. With Albatross knockdown A549 (A1050 and A1160) cells, the aggregated cell population is reduced and free cells are increased. The percentages of single cells in total cells (mean ± SD) are: control, 36.1 ± 3.9; A1050, 52.4 ± 2.8; A1160 cells, 59.4 ± 10.2. n = 4 and P < 0.01. (C) Immunoelectron microscopy of A549 cells with anti-Albatross antibodies. Note that the cytoplasm in the vicinity of AJCs is labeled. TJ, AJ, and DS are indicated. Arrows indicate cell–cell contacts. (D) Quantitative data from C. (E) BC fraction and AJ fraction were immunostained for Albatross with the indicated AJC proteins, PKCζ or Par3. Note that Albatross is well colocalized with them. (F) Immunoblotting of fractions derived from mouse liver: homogenates (left), BC (middle), and AJ (right). Not only Albatross but also Par3 is enriched in line with the concentrations of the indicated AJC components. (G) Immunoprecipitation of A549 cells with anti-Albatross antibodies. Start and IP indicate starting lysates and immunoprecipitates with preimmune (Pre.) and anti-Albatross (αAlb.) antibodies, respectively. Note the Par3 precipitation with Albatross. Among AJC components, ZO-1 also coprecipitated. (H) Immunoprecipitation analysis with tagged Albatross and Par3. Start and IP indicate starting lysates and immunoprecipitates with anti-GFP antibodies, respectively. Left lanes show results for negative controls expressing GFP alone. Par3 was the most precipitated with GFP-Albatross among coexpressed myc-Par3, -Par6, and -PKCλ. Bars: (A) 10 μm; (B) 100 μm; (C) 0.1 μm; (E, BC) 13 μm; (E, AJ) 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Double Staining, Immuno-Electron Microscopy, Labeling, Western Blot, Derivative Assay, Immunoprecipitation, Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Functions of keratins and Albatross–Par3 complexes. (A–C) The amounts of Albatross protein and mRNA were analyzed in both keratin 8 and keratin 18 (K8/18)-introduced SW13 cells. As a control, an empty vector was transfected. As loading controls, α-tubulin and GAPDH were used. Two independent experiments were performed. (A) Immunoblotting. In transiently K8/18-introduced SW13 cells, the amount of Albatross protein is elevated, along with the amount of keratin 18. (B) With stable lines, the same results were obtained. (C) RT-PCR. In K8/18-introduced SW13 cells, the mRNA level of K18 is elevated, but not that of Albatross. β-actin is included as an internal control. (D) Double staining for K8/18 and the indicated proteins: Albatross, AJC components of ZO-1 and afadin, and Par3. (top) In control cells, K8/18 is absent and only limited amounts of Albatross are apparent at cell–cell junctions. In stably K8/18-introduced SW13 cells, Albatross is well localized in cell–cell junctions compared with control cells. (middle and bottom) ZO-1, afadin, and Par3 similarly accumulated at the cell–cell borders in stably K8/18-introduced SW13 cells. (E) Immunostaining of stably K8/18-introduced SW13 cells transfected with control or Albatross siRNA. Note that ZO-1, afadin, and Par3 are reduced at cell–cell borders with knockdown of Albatross. (F) A model for the regulation of AJC and lateral domains with the Albatross–Par3 complex and keratins. Albatross–Par3 complexes regulate the formation of AJC and maintain lateral membrane identity. However, Par3 without Albatross regulates apical structures. Keratins stabilize Albatross, promoting the formation of AJC. Knockdown effects are also indicated. Bars, 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Staining, Stable Transfection, Immunostaining

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 3. Activation of extracellular signal-regulated kinase (ERK), p38 mitogen- activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) in COOH-HBFN-f–stimulated RSF. After incubation with 100 nM COOH-HBFN-f (indicated as HBFN-f) or 2 ng/ml IL-1 for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-pospho-ERK1/2 (ph-ERK1/2), anti-ERK1/2, anti-phospho-p38 MAPK (ph-p38), anti-p38, anti-phospho-JNK (ph-JNK), or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 4. Effects of MAPK inhibitors on MMP production by RSF stimulated with COOH-HBFN-f. After preincubation with PD98059 or SB203580 for 1 hour at the concentrations indicated, RSF were incubated for 48 hours with 100 nM COOH-HBFN-f (indicated as HBFN-f). Conditioned media were analyzed by immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Incubation, Western Blot, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 7. Activation of ERK, p38, and JNK in RSF with 41 integrin ligation with CS-1. After incubation with CS-1 at 10 M for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-ph-ERK1/2, anti-ERK1/2, anti-ph- p38, anti-p38, anti-ph-JNK, or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Ligation, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 8. Effects of MAPK inhibitors on MMP induction by 41 integrin stimulation with CS-1. RSF were pretreated for 1 hour with the indicated concentrations of PD98059 or SB203580 and thereafter stimulated for 48 hours with 10 M CS-1. Conditioned media were analyzed by SDS-PAGE and immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: SDS Page, Western Blot, Derivative Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal weight (A) and umbilical artery serum DHAS (B) and cortisol (C) levels in untreated baboons on days 100 (middle, n = 4) and 165 (late, n = 8) of gestation and on day 165 in animals that were treated daily on days 100–164 with letrozole (0.115 mg/kg body weight per day, n = 8) or letrozole plus estradiol (each at 0.115 mg/kg body weight per day, n = 5). Values indicated by different letter superscripts are different at P < .05 to P < .01 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques:

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Western immunoblot of MC2R (A) and MRAP (B) incubated with primary antibody (Ab), with primary Ab preabsorbed with blocking peptide or without primary Ab in the baboon fetal adrenal gland on day 165 of gestation in untreated and letrozole-treated baboons (n = 2 samples each). Panels C and D show Western immunoblots of MC2R and MRAP in the fetal liver and epididymis on day 165 in an untreated baboon.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Western Blot, Incubation, Blocking Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole on days 100–164 (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R protein expression, quantified by PLA and image analysis/Metamorph software, on day 100 (midgestation, n = 4) and day 165 (late gestation, n = 8) in untreated baboons and on day 165 in animals treated with letrozole (n = 8) or letrozole plus estradiol (n = 5). Each red PLA signal represents a single molecule of MC2R protein detected by primary MC2R antibody tagged with a secondary antibody conjugated to fluorescently labeled oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining with Alexa Fluor 488-conjugated IgG within TZ (A and B) and FZ (C and D) cells is shown in green. Final magnification, ×400 (A–D).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MRAP protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MRAP quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .03 in late vs midgestation.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R-MRAP protein interaction assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R-MRAP interaction quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. For PLA detection of MC2R-MRAP protein interaction, tissue was incubated with a secondary antirabbit PLUS antibody and a secondary antigoat minus antibody conjugated with oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .05 in letrozole-treated animals vs all other groups (ANOVA and Tukey-Kramer multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Software, Incubation, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal liver hepatocyte (A and B) and fetal epidydimal stroma (D and E) MC2R-MRAP interaction assessed by PLA on day 165 of gestation in untreated (A and D) and letrozole-treated (B and E) baboons. α-Smooth muscle actin immunostaining within fibromuscular cells of epidydimal ducts appears in green in panels D and E. Means ± SE of fetal liver (C) and fetal epidydimal stroma (F) MC2R-MRAP interaction were quantified by PLA in baboons untreated (n = 3) or treated with letrozole (n = 3). Final magnification, ×400 in each panel.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: A, Fetal pituitary POMC mRNA expression was quantified by in situ hybridization on days 100 (n = 3) and 165 (n = 7) in untreated baboons and on day 165 in animals treated with letrozole (n = 7) or letrozole plus estradiol (n = 7). B, Fetal (ie, umbilical artery) plasma ACTH levels on days 100 (n = 8) and 165 (n = 18) in untreated baboons and on day 165 in animals treated on days 100–164 with letrozole (n = 14) or letrozole plus estradiol (n = 11). Fetal pituitaries for POMC mRNA assay and blood samples for plasma ACTH assay were obtained from animals of the current study and from a contemporaneous group of baboons from our primate colony. Values indicated by different letter superscripts are different at P < .05 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, In Situ Hybridization

Journal:

Article Title: Extracellular Signal-Regulated Kinase Activates Topoisomerase II? through a Mechanism Independent of Phosphorylation

doi:

Figure Lengend Snippet: Coimmunoprecipitation of endogenous topoisomerase IIα and ERK2 from nuclear extracts. ERK2 was immunoprecipitated from nuclear extracts prepared from nocodazole-treated NIH 3T3 cells; this was followed by immunoblotting to visualize both ERK2 and coimmunoprecipitating topoisomerase (Topo) IIα. Lanes 3 and 4 show increasing amounts of immunoprecipitated ERK2 and coimmunoprecipitated topoisomerase IIα. A small amount of topoisomerase IIα nonspecifically bound to the protein A-(Prot. A)-Sepharose resin (lane 2). Ten percent of the extract volume used for the immunoprecipitations served as a loading control (lane 1).

Article Snippet: For coimmunoprecipitations of endogenous ERK2 and topoisomerase IIα, nuclear extracts from nocodazole-treated NIH 3T3 cells were incubated with 0, 0.2, or 2 μg of anti-ERK2 antibody (C-14; Santa Cruz Biotechnology) for 2 h on ice; this was followed by addition of 20 μl of protein A-Sepharose (Pharmacia) that had been pretreated with BSA at 0.5 mg/ml.

Techniques: Immunoprecipitation, Western Blot