human dsg1  (Cusabio)

Journal: Archives of Dermatological Research

Article Title: Pilot study investigating BP-180 in extracellular vesicles derived from blister fluid of bullous pemphigoid patients

doi: 10.1007/s00403-023-02560-2

Figure Lengend Snippet: Demographics, clinical and immunological characteristics and western blot results of BP patients and controls

Article Snippet: After blocking nonspecific sites with 5% non-fat dry milk (EuroClone, Italy) in Tris Buffered Saline with Tween 20 (TTBS, 20 mM Tris pH 7.5, 500 mM NaCl, 0.05% Tween 20), blot membrane was incubated overnight at 4 °C with specific primary antibodies for: rabbit anti-human BP180 (1:1000, in house), goat anti-human BP230 (1:40, Santa-Cruz) goat anti-human DSG1 (1:1000, R&D), and mouse anti-human CD63 (1:1000 dilution, 10628D, Invitrogen), prepared in 2.5% non-fat dry milk/TTBS.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Microenvironmental network of clonal CXCL13+CD4+ T cells and Tregs in pemphigus chronic blisters

doi: 10.1172/jci166357

Figure Lengend Snippet: Figure 1. Skin tertiary lymphoid structures in chronic blisters of patients with pemphigus. (A) Skin biopsy sample showing tertiary lymphoid structures (TLSs) from a patient with PV (Supplemental Figure 1A, no. 3) stained with H&E and specific antibodies against CD20, CD4, CD138, and PNAd. Scale bar: 100 μm. (B) Schematic of the experiment. (C) Duration of skin blisters in TLS-negative (n = 8) and TLS-positive (n = 23) lesions. Student’s t tests were used to com- pare means for 2 groups. Data are shown as the mean ± SD. **P < 0.005. (D) Representative immunofluorescence staining for DSG-specific B cells (triangles) and plasma cells (arrows). Tissues were stained with CD138 (red), CD20 (blue), rDSG1 or rDSG3 (green), and DAPI (light gray). Dotted lines indicate the margin of TLSs. Scale bar: 50 μm. PF, pemphigus foliaceus; PV, pemphigus vulgaris; rDSG1, recombinant desmoglein 1; rDSG3, recombinant desmoglein 3.

Article Snippet: Tissues were stained overnight at 4°C using the following primary antibodies: mouse anti-human CD20 (L26, Abcam); 6X His-recombinant human DSG1 and 6X His-recombinant human DSG3 (both from Cusabio); goat anti-human CXCL13 (R&D Systems); rabbit anti-human CCL5 (P230E, Thermo Fisher Scientific); rabbit anti-human CD138 (EP201), rabbit anti-human FDC (CNA.42), and mouse anti-human CD8 (C8/144B) (all from Cell Marque); rat anti-human CD4 (YNB 46.1.8, Santa Cruz); and mouse anti-human HLA-DR (L243, BioLegend).

Techniques: Staining, Immunofluorescence, Clinical Proteomics, Recombinant

Journal: bioRxiv

Article Title: Aryl Hydrocarbon Receptor Suppresses Eosinophilic Esophagitis Responses through OVOL1 and SPINK7

doi: 10.1101/2023.05.17.541192

Figure Lengend Snippet: qPCR analysis of CYP1A1 (A) and SPINK7 (B) expression after stimulation with the indicated stimuli with or without GNF351. A and B, red dashed line represents the basal expression of CYP1A1 and SPINK7 in UT cells respectively. C. Representative images of co-immunofluorescence of desmoglein1 (DSG1, green), OVOL1 (pink) and DAPI (blue) after 0, 1 or 18 hrs of stimulation with FICZ (1 µm), ITE (1 µm), or Omeprazole (10 µm). D. Representative western blot of OVOL1 expression in cell fractions. LaminB1 represents a nuclear loading control and HSP90 represents a cytosolic loading control. E. Heatmap representing TPM values of genes that are significantly altered by FICZ treatment (p adjusted < 0.05). F. Gene ontology (GO) enrichment analyses of genes that are induced by FICZ treatment. P value for GO analysis was calculated by an ANOVA test. G. Genes differentially expressed (fold change > I2I) in EPC2 cells following 18 hrs of FICZ stimulation (AHR transcriptome) compared with untreated cells (left column; AHR) compared to the EoE transcriptome (right column; EoE) obtained from RNA sequencing of esophageal biopsies (n = 6 control patients [Control] and n = 10 patients with active EoE [EoE] as described in ). Venn diagrams compare the genes of the AHR transcriptome and the EoE transcriptome. Heatmap represents the fold change of genes that overlapped between the FICZ transcriptome and the EoE transcriptome. H. GO analysis depicting cellular components of the overlapping genes. The p value for GO analysis was calculated by ANOVA. UT, untreated.

Article Snippet: Slides were blocked in 1X phosphate-buffered saline (PBS) with 10% goat serum for 1 h followed by a 1-h incubation at room temperature in the following primary antibodies: rabbit anti-human OVOL1 (Sigma Aldrich) and mouse anti–human DSG1 (Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Western Blot, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Aryl Hydrocarbon Receptor Suppresses Eosinophilic Esophagitis Responses through OVOL1 and SPINK7

doi: 10.1101/2023.05.17.541192

Figure Lengend Snippet: A . Representative images of co-immunofluorescence of desmoglein1 (DSG1, green), OVOL1 (pink), and DAPI (blue) stain in OVOL1-overexpressing cells that were either left untreated (UT) or treated over night with IL-4 or IL-13 (100 ng/mL) with or without FICZ (1 µm). Promoter activity in lysates triple-transfected with firefly vector and either SPINK7 -nLUC or nLUC and either OVOL1 or a control plasmid. Cells were either left untreated or treated with 1 µm FICZ, with or without IL-4 (B) or IL-13 (C). D. Representative images of co-immunofluorescence of DSG1 (pink), OVOL1 (cyan), and DAPI (blue) stain in cells that were differentiated in the ALI model. Cells were either left untreated or treated with FICZ (1 µm), IL-4 or IL-13 (100 ng/mL), or IL-4 (100 ng/mL) with FICZ (1 µm). mRNA expression of SPINK7 (E) , CYP1A1 (F) , or CCL26 (G) normalized to GADPH in cells that were either left untreated or stimulated with IL-13 (100 ng/mL) and/or FICZ (1 µm). H. Principal component analysis using a permanova weighted test of dysregulated genes by RNAseq data from cells that were either left untreated or stimulated with IL-13 (100 ng/mL) and/or FICZ (1 µm). I. Overlap between the IL-13 transcriptome (IL-13 compared to UT) and IL-13 and FICZ transcriptome (IL-13 + FICZ compared to UT). J. The fold change of IL-13 treatment compared to UT and the fold change of IL-13 + FICZ treatment compared to UT of top upregulated genes in the IL-13 transcriptome.

Article Snippet: Slides were blocked in 1X phosphate-buffered saline (PBS) with 10% goat serum for 1 h followed by a 1-h incubation at room temperature in the following primary antibodies: rabbit anti-human OVOL1 (Sigma Aldrich) and mouse anti–human DSG1 (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Activity Assay, Transfection, Plasmid Preparation, Control, Expressing

Journal: bioRxiv

Article Title: Aryl Hydrocarbon Receptor Suppresses Eosinophilic Esophagitis Responses through OVOL1 and SPINK7

doi: 10.1101/2023.05.17.541192

Figure Lengend Snippet: A. Western blot analysis of OVOL1, DSG1, and GAPDH expression in differentiated EPC2 cells that were either left untreated (UT) or stimulated with IL-13 (100 ng/mL) for 48 h. The graphs on the right show quantification of OVOL1 and DSG1 relative to GAPDH with or without IL-13 treatment from paired UT/IL-13 samples from 6 independent experiments. B. qPCR analysis of OVOL1 and DSG1 mRNA expression in differentiated EPC2 cells that were either left untreated or stimulated with IL-13 (100 ng/mL) for 48 h; expression is normalized to GAPDH . C. Heatmap depicting the relative expression of the indicated genes in epithelial clusters on the basis of single-cell RNA sequencing data of dispersed cells from esophageal control biopsies. D. Western blot analysis of OVOL1 and calpain 14 expression in differentiated EPC2 cells with inducible expression of CAPN14 expression. CAPN14 is fused to a flag tag and is induced by doxycycline (Dox) treatment. qPCR analysis of OVOL1 (E) and SPINK7 (F) in differentiated EPC2 with inducible expression of CAPN14 expression of CAPN14 ; expression is normalized to GAPDH . G. Western blot analysis of OVOL1 in GFP-overexpressing or CAPN14-GFP– overexpressing cells with or without IL-13 treatment. GAPDH was used as a loading control. Anti-GFP was used for detection of GFP and CAPN14-GFP. H. Western blot analysis of protein lysates that were incubated for the indicated times with recombinant OVOL1 protein (100 ng) with or without calpastatin or A1AT. The graph on the right side represents the mean values ± SEM of the measured fluorescent signal of the degradation products of OVOL1, calculated as percent from the measured fluorescent signal of the full length of OVOL1 at time 0, from 3 independent experiments. I. Western blot analysis of recombinant human OVOL1-GST (60 ng) that was either left untreated or incubated with cytoplasmic protein fractions (C) or nuclear protein fractions (N) for the indicated times. The graph on the right is a quantification of OVOL1 band intensity (O.D). NS, not significant; UT, untreated.ht is a quantification of OVOL1 band intensity (O.D).

Article Snippet: Slides were blocked in 1X phosphate-buffered saline (PBS) with 10% goat serum for 1 h followed by a 1-h incubation at room temperature in the following primary antibodies: rabbit anti-human OVOL1 (Sigma Aldrich) and mouse anti–human DSG1 (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, RNA Sequencing, Control, FLAG-tag, Incubation, Recombinant