Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: (A) Timeline of experimental manipulations used in these studies. (B) Map outlining the genetic maps of the various retroviral vectors used in these studies. Each viral vector has the coding sequence of one or two genes in its multiple cloning site, which sits 5’ (upstream) of an IRES-eGFP sequence. The vectors are identified in name by their respective inserts: empty vector, Sox2 , Pax6 , and SEP ( Sox2-E2A-Pax6 ). (C) Western blot performed on lysates from HEK293T cells transfected with the vectors, confirming protein production of each gene product at the expected molecular weights when stained with antibodies against Pax6, Sox2 and GFP. Lane 1: pLIA-Pax6, lane 2: pLIA-SEP, lane 3: pLIA-Sox2, lane 4: pLIA-GFP, lane 5: untransfected HEK cells, lane 6: molecular weight standards.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Plasmid Preparation, Sequencing, Clone Assay, Western Blot, Transfection, Staining, Molecular Weight

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Derivative Assay, Sequencing, Affinity Purification, Recombinant, Purification, Chromatography

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: (Col. A) Sox2, (Col. B) Pax6, (Col. C) Ascl1 (Mash1), and (Col. D) TuJ1 (neuron-specific tubulin) immunostaining in control mouse OE and at 1, 2, 4 and 7 days post MeBr-lesion (dpl). Note the reappearance and patterning of transcription factor expression. CK14 labeling marks the horizontal basal cells in the uninjured epithelium and the cells that derive from them at the early stages in the reconstitution of the epithelium, including ones that transition into Sox2/Pax6 (+)/CK14 (-) GBCs. Regeneration of olfactory sensory neurons is marked by the expression of neuron-specific tubulin (marked by Tuj1 staining). The vast majority of spared cells express both Pax6 and Sox2 up through 4 dpl. By this time Ascl1-expressing GBCs have made a limited reappearance and a few Tuj1 (+), neurons have emerged. Dashed lines mark the basal lamina. Scale bar in (A, uninjured) equals 10 μm and applies to all panels.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Immunostaining, Expressing, Labeling, Staining

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: Methods for Antibody Staining used in this study.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Staining

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: (A) A proneural bHLH cascade operates during olfactory neurogenesis and defines a hierarchy of GBCs as they progress from transit amplifying (TA) progenitor cells into differentiated neurons, by way of two immediate neural precursor (INP) stages. (B-M) At 8-days post-infection (9-days post-lesion), tissue was immunolabeled for Ascl1, Neurog1, and NeuroD1 along with GFP in clones derived from EV (B, F, J), Sox2 (C, G, K), Pax6 (D, H, L) and SEP (E, I, M) RVV-transduction. Hollow arrows mark GFP (+) cells within a given clone that co-express Ascl1, Neurog1, or NeuroD1. In contrast, solid arrows mark non-transduced, i.e., GFP-lacking, cells that express these factors. Dashed lines mark the basal lamina and scale bar in K corresponds to 20 μm in panels B-M. (N-P) Quantitative analysis of the effect of the various transduction conditions on the percentage of cells in a clone that express Ascl1 (N), Neurog1 (O) and NeuroD1 (P). For the graph, the percentage of cells marked by joint expression of the individual transcription factor and GFP within each clone are graphed in a box-and-whiskers scatter plot; the median number of each distribution is marked by the horizontal black line within the box. For Ascl1+ GBCs, the median percentages for EV vs. Sox2 vs. Pax6 vs. SEP are 0, 36, 0, 20, respectively. For Neurog1+ GBCs, the median percentages were 0, 37, 17, 25, respectively. Finally, for NeuroD1+ GBCs, the median values were 0, 50, 33, 19, respectively. Multiple pair-wise comparisons (horizontal line) were found to be statistically significant using Dunn’s method (* p < 0.05) following the demonstration of overall significance using Kruskal-Wallis One-Way ANOVA tests. (Q) Percentage of clones that contain cells positive for labeling with the bHLH factors. To compare percentages statistically, data were first arcsin transformed prior to normality testing, standard parametric ANOVA, and post-hoc Holm-Sidak pair-wise testing (**p < 0.01). Asterisks signify comparisons to EV, except for the comparison in number of Ascl1-conatining clones between Sox2 and Pax6 vectors, which is designated by the orthogonal lines.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Infection, Immunolabeling, Clone Assay, Derivative Assay, Transduction, Expressing, Labeling, Transformation Assay

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: (A-H) Tissues harvested 3 weeks after EV , Pax6 , SEP and Sox2 RVV-transduction were stained with GFP, along with PGP9.5 (a neuronal marker) to illustrate the range in outcomes for each condition. Cell identity among clones was then determined relative to PGP9.5 staining. GFP-labeled cells were identified as sustentacular cells (Sus) when their cell bodies were found superficial to the band of PGP9.5 (+) OSNs, while GFP-labeled cells were identified as basal cells if they were deep to the band of neurons. GFP (+) cells were identified as duct/gland cells (D/G) when they were flattened, oriented as a chain of cells along the apical-basal axis, and/or extended deep to the basal lamina into the lamina propria (cf. ). GFP (+) cells were identified as OSNs, if they co-labeled with PGP9.5. (E-H) CD54 was used to immunostain HBCs along with PGP9.5. (E-H) There is substantial variation in clonal composition. (E) The arrow indicates a mixed clone composed of OSNs and a large group of Sus cells atop the neurons. (F) The arrow indicates a Sus-only clone where the GFP-labeled cells sit atop the neurons. (G) The solid arrow with asterisk points to gland cells located deep to the HBCs and basal lamina. (H) Another example of a mixed clone composed of Sus cells (arrow) and neurons (hollow arrow with asterisk). Dashed lines mark the basal lamina (A-H) and the scale bar in (H) corresponds to 20 μm and applies to all of the panels. (I) For the various cell types, the data from each and every clone was used to generate a box-and-whiskers scatter plot; the median for each distribution is marked by the horizontal black line within the box. In the order shown in the graph they are: for basal cells– 1, 1, 1, 0; for OSNs– 8, 2, 0, 3; for Sus cells– 3, 2, 0, 1; for D/G (duct/gland cells)– 0, 0, 0, 0, respectively. Note the break in the ordinate of the graph to accommodate those clones that contain a markedly greater number of neurons, which occur exclusively with Sox2 transduction. Because all of the datasets shown here failed the Shapiro-Wilk test for normality, a Kruskal-Wallis One-Way ANOVA on Ranks was used, followed by Dunn’s Method for pairwise multiple comparisons. (J) For the group of clones as a whole, the percentage of clones that contain neurons for each form of transduction are indicated in the bar graph, and multiple pairwise comparisons (indicated by the horizontal lines) are significantly different (Fisher Exact test with Holm-Sidak multiple comparison correction, * p < 0.05).

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Transduction, Staining, Marker, Clone Assay, Labeling

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: (A-D) GFP expression in representative whole mounts, demonstrating typical clone size following transduction with EV , Sox2 , Pax6 , and SEP retroviral vectors, respectively. Scale bar in (D) corresponds to 50 μm and applies to all photos. (E) The range in cell number per clone is depicted as a box-and-whiskers scatter plot, and the median clone size is indicated by the horizontal black line within the box. The number of clones analyzed were as follows: EV – 49, Sox2 –111, Pax6 –150, and SEP – 165. Non-parametric ANOVA was statistically significant (Kruskal-Wallis, p < 0.05) as were post-hoc pairwise comparisons using Conover-Iman tests of multiple comparisons using rank sums; * designates p < 0.05.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Expressing, Transduction, Clone Assay

Journal: PLoS ONE

Article Title: Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium

doi: 10.1371/journal.pone.0155167

Figure Lengend Snippet: Clones from tissue harvested 3-weeks after Cre RVV-transduction of bigenic R26 fl(stop)-LacZ ; Sox2 flox/flox mice were immunolabeled for the neuronal marker PGP9.5 (A) or the HBC marker CD54 (B), along with Pax6 and GFP. In clones containing GFP (+)/ PGP9.5 (-) Sus cells (A), situated above the neural strata (arrows), Pax6 expression remains indistinguishable from surrounding untransduced Sus cells. In clones containing GFP (+)/CD54 (+) HBCs (B), Pax6 levels remain indistinguishable from untransduced HBCs (arrows). Arrowheads mark the basal lamina and scale bar corresponds to 20 μm and applies to all panels.

Article Snippet: Mouseα Pax6 , Developmental Studies Hybridoma Bank , Recombinant protein corresponding to N-terminus of chicken Pax6, amino acids 1–223.

Techniques: Clone Assay, Transduction, Immunolabeling, Marker, Expressing

Journal: Nature Communications

Article Title: Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence

doi: 10.1038/s41467-020-16337-y

Figure Lengend Snippet: a BCA (protein concentration) of circulating EVs from healthy donors ( h EVs) and P. vivax patients ( Pv EVs) ( n = 10, individual samples). Data show mean ± SD. Two-sided, Mann–Whitney test, * p = 0.0232 (GraphPad). b P. vivax proteins identified by different unique peptides (sequences below the description of corresponding proteins). UniProtKB accession numbers and gene name corresponding to the P. vivax PvP01 strain are shown. c Western blot analysis of Pv EVs obtained from different individual patients (Pv1-7 and Pv10) and human donors (H1 and H3) using anti P. vivax MSP3.1 (upper membrane) and PHISTc (bottom membrane) antibodies. MSP3.1 and PHISTc recombinant truncated-proteins fused to GST, were used as positive controls. Molecular weight in kDaltons (kDa) is shown to the left. M: molecular size marker. Image representative of three independent experiments. d Nanoparticle tracking analysis (NTA) profile (size [nm] versus concentration [particles/mL]) of pooled h EVs and Pv EVs was analysed using NanoSight LM10-12. Table shows mean of three measurement of pooled h EVs and Pv EVs quantified by NTA (particles concentration) and BCA (protein concentration). e Beads-based flow cytometry analysis of pooled h EVs and Pv EVs using six EV markers (CD9, CD63, CD81, GAL3, CD5L and CD71). Data show median fluorescence intensity (MFI) of each antibody and control antibodies (rabbit and mouse-isotype) ± SD (technical replicates, n = 3). Unpaired and two-sided, t -test ** p = 0.0032 (CD63), *** p = 0.0006 (CD81, CD71), **** p < 0.0001 (CD9) (GraphPad). Source data are provided as a Source data file and Supplementary Data , and .

Article Snippet: Further, cells were stained using the following antibodies: CD90-PE/Cy7 [5E10] (Biolegend, Cat#328124) 1/100; CD44-FITC [KM201] (Abcam Cat#ab25340) 1/100; CD54-Alexa Fluor® 488 [HCD54] (Biolegend, Cat#322714) 1/400; CD71-PE [AC102] (Miltenyi Biotec, Cat#130-091-728) 1/400; CD45-PerCP [2D1] (BD Biosciences, Cat#345809) 1/50.

Techniques: Protein Concentration, MANN-WHITNEY, Western Blot, Membrane, Recombinant, Molecular Weight, Marker, Concentration Assay, Flow Cytometry, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Enzyme-linked Immunospot

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Expressing

Journal: Endocrinology

Article Title: Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice.

doi: 10.1210/en.2012-2080

Figure Lengend Snippet: Figure 3. A, Presence of TLR4 in podocytes and adipocytes. Representative RT-PCR results show 109-bp products; MM, 100-bp molecular weight marker. B, Representative Western blot of TLR4 in intrinsic renal cells. C, Densitometric analysis of Western results. Experimental cells were cultivated under HG conditions (30 mM of glucose) for 72 hours and harvested for protein extraction. MC, mesangial cells; PTC, proximal tubule cells. Data are shown as mean SEM; *, P .05; ***, P .001 vs MC.

Article Snippet: Proteins were transferred onto a polyvinylidene difluoride membrane, and the membrane was hybridized in blocking buffer overnight at 4°C with goat polyclonal anti-TLR4 antibody (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-NF-kB (p65) antibody (1:1000; Cell Signaling Technology, Danvers, Massachusetts), mouse monoclonal anti-IkB antibody (1:1000, Cell Signaling Technology), goat polyclonal anti-TRAF6 antibody (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-Sp-1 (specificity protein-1) antibody (1:1000, Millipore), rabbit polyclonal Nox4 antibody (Novus Biologicals, Littleton, Colorado), rabbit polyclonal anti-PAI-1 antibody (1:500; Santa Cruz Biotechnology), rabbit polyclonal antitype IV collagen antibody (1:500; Santa Cruz Biotechnology), rabbit polyclonal anti-TGF- 1 antibody (1:200; Santa Cruz Biotechnology), goat polyclonal antiSmad2/3 antibody (1:200; Santa Cruz Biotechnology), rabbit polyclonal phospho-Smad2 antibody (1:200; Cell Signaling Technology), rabbit polyclonal phospho-Smad3 (1:200; Cell Signaling Technology), and mouse monoclonal anti- -actin antibody (1:5000, Sigma-Aldrich).

Techniques: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Western Blot, Protein Extraction

Journal: Endocrinology

Article Title: Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice.

doi: 10.1210/en.2012-2080

Figure Lengend Snippet: Figure 6. Expression of TLR4 and the Inhibitory Effect of GIT27 in Experimental Animals A, Representative Western blots of specificity protein 1 (SP-1), NF-B (p65), TLR4, IB, and Nox4 in renal cortical tissues. B, Densitometric analysis of Western results. Data are shown as mean SEM; **, P .01; ***, P .001 vs db/m; #, P .05; ##, P .01; ###, P .001 vs control db/db.

Article Snippet: Proteins were transferred onto a polyvinylidene difluoride membrane, and the membrane was hybridized in blocking buffer overnight at 4°C with goat polyclonal anti-TLR4 antibody (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-NF-kB (p65) antibody (1:1000; Cell Signaling Technology, Danvers, Massachusetts), mouse monoclonal anti-IkB antibody (1:1000, Cell Signaling Technology), goat polyclonal anti-TRAF6 antibody (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-Sp-1 (specificity protein-1) antibody (1:1000, Millipore), rabbit polyclonal Nox4 antibody (Novus Biologicals, Littleton, Colorado), rabbit polyclonal anti-PAI-1 antibody (1:500; Santa Cruz Biotechnology), rabbit polyclonal antitype IV collagen antibody (1:500; Santa Cruz Biotechnology), rabbit polyclonal anti-TGF- 1 antibody (1:200; Santa Cruz Biotechnology), goat polyclonal antiSmad2/3 antibody (1:200; Santa Cruz Biotechnology), rabbit polyclonal phospho-Smad2 antibody (1:200; Cell Signaling Technology), rabbit polyclonal phospho-Smad3 (1:200; Cell Signaling Technology), and mouse monoclonal anti- -actin antibody (1:5000, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Control

Journal: Infection and Immunity

Article Title: EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

doi: 10.1128/iai.00072-08

Figure Lengend Snippet: FIG. 2. Alignment of the EspF orthologs with human N-WASP and WIP. (A) Phylogram of EspF sequences and of WASP, N-WASP, WIP, and RickA genes currently available in public databases. (B) Amino acid sequence alignment of EspF orthologs, WIP, and N-WASP. Two relevant portions are shown and the positions of the potential profilin-, G-actin-, and Arp2/3-binding motifs are indicated over the amino acid sequences.

Article Snippet: Membranes were then probed with antibodies directed against occludin (mouse monoclonal antibody, 1:200), claudin (rabbit polyclonal antibody, 1:200), ZO-1 (rabbit polyclonal antibody, 1:200), ZO-2 (rabbit polyclonal antibody, 1:200; Zymed Laboratories, Inc.), profilin (goat polyclonal antibody, 1:100); Arp-2 (goat polyclonal antibody, 1:100), WASP (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Inc.), EspF (mouse monoclonal antibody, 1: 200; described above), and actin (mouse monoclonal, 1:200; prepared in our laboratory).

Techniques: Sequencing, Binding Assay

Journal: Infection and Immunity

Article Title: EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

doi: 10.1128/iai.00072-08

Figure Lengend Snippet: FIG. 3. EspF binds N-WASP, Arp2/3, profilin, and actin. (A) Co- immunoprecipitation of N-WASP, Arp2/3, and profilin by anti-EspF antibody. RK13 cells were infected with wild-type E22 at various times. After infection, cells were fractionated and the soluble fraction was immunoprecipitated with an anti-EspF antibody. Immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose mem- brane, and probed with antibodies against N-WASP, Arp2, and profilin by Western blotting (WB). Soluble fractions plus protein A-agarose (CytosolProtA) and anti-EspF antibody plus protein-A-agarose (- EspFProtA), as well as infection at time zero, were used as negative controls. MWM, molecular weight marker. (B) Coimmunoprecipita- tion of actin and EspF. Five-microgram portions of purified actin and recombinant EspF-His were mixed. The mix was subjected to immu- noprecipitation by using anti-EspF or anti-actin antibodies. As a neg- ative control, purified EspF was mixed with commercial Hsc70 and the immunoprecipitation was performed with anti-EspF antibodies. Addi- tionally, soluble fractions from infected cells were mixed with 5 g of actin, subjected to immunoprecipitation, and then analyzed by West- ern blotting as indicated above. IP, immunoprecipitation.

Article Snippet: Membranes were then probed with antibodies directed against occludin (mouse monoclonal antibody, 1:200), claudin (rabbit polyclonal antibody, 1:200), ZO-1 (rabbit polyclonal antibody, 1:200), ZO-2 (rabbit polyclonal antibody, 1:200; Zymed Laboratories, Inc.), profilin (goat polyclonal antibody, 1:100); Arp-2 (goat polyclonal antibody, 1:100), WASP (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Inc.), EspF (mouse monoclonal antibody, 1: 200; described above), and actin (mouse monoclonal, 1:200; prepared in our laboratory).

Techniques: Immunoprecipitation, Infection, SDS Page, Western Blot, Molecular Weight, Marker, Recombinant, Control

Journal: Animal : an international journal of animal bioscience

Article Title: The GH/IGF1 axis in the kidney of young goats fed a protein-reduced diet.

doi: 10.1016/j.animal.2023.100897

Figure Lengend Snippet: Fig. 2. Western blot analysis and semi-quantification of (A) growth hormone receptor (GHR) protein, (B) IGF1 (IGF1R) protein, (C) insulin receptor (INSR) protein, (D) Janus kinase 2 (JAK2) protein, (E) suppressor of cytokine signalling 2 (SOCS2) protein, (F) suppressor of cytokine signalling 3 (SOCS3) protein, (G) signal transducers and activators of transcription 1 (STAT1) protein, (H) signal transducers and activators of transcription 3 (STAT3) protein, and (I) signal transducers and activators of transcription 5B (STAT5B) protein expression in the renal cortex of young goats fed a control diet (N + ) or a reduced-protein diet (N-). M, molecular weight marker. Values are means with their SEM represented by vertical bars. *Significant differences between the two groups were revealed by an unpaired Student’s t-test (P < 0.05); **P < 0.01; ****P < 0.001.

Article Snippet: GHR, JAK2, SOCS2, SOCS3, STAT1, STAT3, STAT5B, IGF1R and INSR protein were detected by treating the membranes overnight at 4 C with anti-GHR antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany), anti-JAK2 antibody (Cell Signalling), anti-SOCS2 antibody (Cell Signalling), anti-SOCS3 antibody (Cell Signalling), anti-STAT1 antibody (Cell Signalling), antiSTAT3 antibody (Cell Signalling), anti-STAT5B antibody (GeneTex Inc., Irvine, CA, USA), anti-IGF1R antibody (Cell Signalling) or with anti-INSR antibody (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Control, Molecular Weight, Marker