mouse monoclonal anti dsg2 antibodies  (Bio-Rad)

Journal: bioRxiv

Article Title: Dsg2 truncation causes a lethal barrier breakdown in mice

doi: 10.1101/2024.07.01.601522

Figure Lengend Snippet: (A) Schematic illustrating the strategy for generation of Dsg2 MUT mice. (B) Dsg2 protein level in murine colon samples was determined by western blotting using a Dsg2 antiserum. Dsg2 protein level in Dsg2 MUT mice is reduced and a truncated band is visible. (C) Immunofluorescence staining against Dsg2 in colon tissue reveals diminished Dsg2 immunostaining in enterocytes of Dsg2 MUT mice. Scale bar: 10 µm. (D) A photograph of 10 days old wild-type and Dsg2 MUT mice (E, F) Body size (E) and total body weight (F) of Dsg2 MUT mice are significantly reduced at 10 days of age compared to wild-type mice. (G) Kaplan-Meier Plot on the survival rate of Dsg2 MUT mice and wild-type litters. Dsg2 MUT mice start to die at postnatal day 7 and do not survive longer than day 14. (H) Representative photographs of 10 days old mice after sacrifice with opened peritoneum. Arrows indicate gas filled regions predominantly in distal small intestine and colon of Dsg2 MUT mice. (I, J) Length of small (I) but not large intestine (J) is smaller in Dsg2 MUT mice than in wild-type mice.

Article Snippet: The study was performed using antibodies against Dsg2 (Cat. No. 610121, 1:100, Progen, Heidelberg, Germany, or Cat. No. BM5016, 1:20, Origene, Rockville, MD, USA), E-Cadherin (Cat. No. 610181, 1:100, BD Biosciences, Heidelberg, Gemany), Occludin (Cat. No. 40-4700, 1:100, Thermo Scientific, Waltham, MA, USA), Claudin 2 (Cat. No. 51-6100, 1:100, Thermo Scientific, Waltham, MA, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Dsg2 truncation causes a lethal barrier breakdown in mice

doi: 10.1101/2024.07.01.601522

Figure Lengend Snippet: (A) Images obtained during consecutive colonoscopies in 2015, 2017 and 2019, which reveal mucosal inflammation with ulcerations and progressive loss of vascularity. (B) Biopsy specimen taken from the ascending colon with hematoxylin and eosin staining (left panel, asterisks) shows inflammatory infiltrations, and immunostaining (right panel) for the extracellular domain of Dsg2 reveals fragmented Dsg2 localization. (C) Schematic illustrating the 15 coding exons of the human DSG2 gene and the Dsg2 protein. The novel pathogenic mutation leads to a truncation of the terminal part of the cytoplasmic domain.

Article Snippet: The study was performed using antibodies against Dsg2 (Cat. No. 610121, 1:100, Progen, Heidelberg, Germany, or Cat. No. BM5016, 1:20, Origene, Rockville, MD, USA), E-Cadherin (Cat. No. 610181, 1:100, BD Biosciences, Heidelberg, Gemany), Occludin (Cat. No. 40-4700, 1:100, Thermo Scientific, Waltham, MA, USA), Claudin 2 (Cat. No. 51-6100, 1:100, Thermo Scientific, Waltham, MA, USA).

Techniques: Staining, Immunostaining, Mutagenesis

Journal: bioRxiv

Article Title: Dsg2 truncation causes a lethal barrier breakdown in mice

doi: 10.1101/2024.07.01.601522

Figure Lengend Snippet: (A) Western blot analysis confirming the reconstitution of DLD-1 enterocytes defective for Dsg2 and Dsc2 (DLD-1 ΔDSC2 ΔDSG2) with the Dsg2 truncated form at Leucin 772 (DSG2Leu772*-EGFP) and Dsg2 (DSG2-EGFP) in contrast to the transfection with the control vector pEGFP-EV (n=4). (B) Immunofluorescence analysis after transfection of DLD-1 ΔDSC2 ΔDSG2 cells with the indicated plasmids. Co-staining with the Dsg2 autoantibody revealed successful and specific reconstitution of Dsg2. DSG2-EGFP and its truncated form is localized at cell borders. However, in cells that were reconstituted with DSG2Leu772*-EGFP a reduction of Dsg2 staining and localization on the cell borders were less continuous in comparison to the reconstitution with DSG2-EGFP. No specific staining was observed with the control vector pEGFP-EV. DAPI-staining proved the number and vitality of cells (n=3). (C) Representative images of FRAP of DLD-1 ΔDSC2 ΔDSG2 cells reconstituted with DSG2-EGFP and with DSG2Leu772*-EGFP revealing that the immobile fraction of truncated Dsg2 was significantly reduced in comparison to DSG2-EGFP. The white box highlights the bleached area. (D) Quantification of the average fraction of immobile DSG2Leu772*-EGFP and DSG2-EGFP is shown in Panel D (*p < 0.05; error bars represent SEM; N=4).

Article Snippet: The study was performed using antibodies against Dsg2 (Cat. No. 610121, 1:100, Progen, Heidelberg, Germany, or Cat. No. BM5016, 1:20, Origene, Rockville, MD, USA), E-Cadherin (Cat. No. 610181, 1:100, BD Biosciences, Heidelberg, Gemany), Occludin (Cat. No. 40-4700, 1:100, Thermo Scientific, Waltham, MA, USA), Claudin 2 (Cat. No. 51-6100, 1:100, Thermo Scientific, Waltham, MA, USA).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: Dsg2 truncation causes a lethal barrier breakdown in mice

doi: 10.1101/2024.07.01.601522

Figure Lengend Snippet: (A) Levels of 4kDa FITC dextran fluorescent concentration in medium, in which a sealed, dye-filled intestinal segment was immersed, increased over time in Dsg2 MUT mice but not in wild-type mice, indicating a higher intestinal permeability Dsg2 MUT mice. (B) Slope of this increase in fluorescent intensity as shown in (A) corresponds to the apparent permeability (*p ˂ 0.05; n = 5). (C) Hematoxylin and eosin staining of colonic fragments. Notably, Dsg2 MUT mice exhibit no gross mucosal damage, but intestinal edema as indicated by arrows. Scale bar: 50 µm. (D) Electron microscopy revealed ultrastructural morphological alterations, including asymmetric appearance of the desmosomes and a widened intercellular space (E) Representative Western blotting analysis of protein expression of plakoglobin (Pg), occludin (Ocln), E-cadherin (Ecad) and claudin 2 (Cldn2) in colon tissue samples of Dsg2 MUT and wild-type mice. (F) Quantification of (E) The protein levels of claudin 2 and occludin are significantly increased in Dsg2 MUT mice (*p ˂ 0.05; n ≥ 4). (G) Accompanying immunostaining of colon tissue against claudin 2 and E-Cadherin in Dsg2 MUT mice and wild-type litters. Scale bar: 10 µm.

Article Snippet: The study was performed using antibodies against Dsg2 (Cat. No. 610121, 1:100, Progen, Heidelberg, Germany, or Cat. No. BM5016, 1:20, Origene, Rockville, MD, USA), E-Cadherin (Cat. No. 610181, 1:100, BD Biosciences, Heidelberg, Gemany), Occludin (Cat. No. 40-4700, 1:100, Thermo Scientific, Waltham, MA, USA), Claudin 2 (Cat. No. 51-6100, 1:100, Thermo Scientific, Waltham, MA, USA).

Techniques: Concentration Assay, Permeability, Staining, Electron Microscopy, Western Blot, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Dsg2 truncation causes a lethal barrier breakdown in mice

doi: 10.1101/2024.07.01.601522

Figure Lengend Snippet: (A) Enhanced Volcano plot of upregulated and downregulated genes from neonatal mice colon. (n = 5/genotype). log 2 FC ≥ 1 or ≤ −1 is considered significant. (B) mRNA expression levels for significantly upregulated or downregulated genes relevant for inflammation and immune response (FDR ≤ 0.05). (C) qRT-PCR data showing mRNA expression levels of S100A8, S100A9 and Il17ra in murine colon tissue. Gene expression of the cytokines was calculated according the ΔΔCT method and normalized to GAPDH (n = 5, **p < 0.005). (D) Go biological process gene ontology analysis depicting the number of upregulated genes involved in IL-17 regulation from Dsg2 MUT mice. Values in parentheses represent the number of genes contained within each category. (E) Venńs diagram representing the number of significant and non-significant genes with a log 2 FC ≥ 1 or ≤ −1 from Dsg2 MUT mice and Dsg2-deficient human patients. Overlapping areas show the amount of common differentially regulated genes in both species. (F) Go biological process analysis depicting the number of upregulated genes involved in IL-17 and IL-23 regulation from Dsg2-deficient patients. (G) List of common genes differentially regulated in mouse and human relevant for IL-17 signalling.

Article Snippet: The study was performed using antibodies against Dsg2 (Cat. No. 610121, 1:100, Progen, Heidelberg, Germany, or Cat. No. BM5016, 1:20, Origene, Rockville, MD, USA), E-Cadherin (Cat. No. 610181, 1:100, BD Biosciences, Heidelberg, Gemany), Occludin (Cat. No. 40-4700, 1:100, Thermo Scientific, Waltham, MA, USA), Claudin 2 (Cat. No. 51-6100, 1:100, Thermo Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: Detection of antibodies against ICD proteins in human left ventricular tissue by immunofluorescence analysis. Human left ventricular tissue was incubated with respective AC-IgGs and Control-IgGs as indicated in the figure. N-CAD was used as a marker for ICDs, and presence of anti-ICD proteins was defined when there was an overlap of IgG staining with N-CAD (white arrows). The yellow arrow in control-IgG shows no positive staining of ICDs. Anti-DSG2 antibody was used to show the presence of DSG2 in the cardiac tissue used. AC patients were grouped into DPC (harboring desmoplakin gene mutations) and ARVC (harboring plakophilin2 gene mutations). Images represent immunofluorescence analysis of 3 repeats. Scale bar: 10 µm.

Article Snippet: As a positive control, two different anti-human DSG2 antibodies (DSG2-Origene, #BM5016; DSG2-Abcam, #ab14415) were used at 1:1000 dilutions in blocking buffer.

Techniques: Immunofluorescence, Incubation, Control, Marker, Staining

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: In vitro DSG2 and N-CAD Cleavage assays A. Human DSG2-Fc protein (200ng/lane) or B. N-CAD-Fc protein (200 ng/lane) was incubated with respective IgGs from AC patients (AC1-AC15), water (H 2 O), healthy control IgG (IgG), PV-IgG and AK23 (murine pemphigus monoclonal anti-DSG3 antibody, as a negative control) for 4 hours, without (C) and with protease inhibitor (I, cOmplete™) and Western blot analysis was performed. A representative image of 3 experimental repeats is shown. # Indicates the cleaved fragments. * Indicates mouse IgG heavy chain.

Article Snippet: As a positive control, two different anti-human DSG2 antibodies (DSG2-Origene, #BM5016; DSG2-Abcam, #ab14415) were used at 1:1000 dilutions in blocking buffer.

Techniques: In Vitro, Incubation, Control, Negative Control, Protease Inhibitor, Western Blot

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: Homophilic DSG2 and N-CAD interaction probabilities measured by AFM A . Schematic presentation of an AFM interaction experiment in cell-free conditions. Recombinant DSG2/N-CAD extracellular domain containing proteins tagged with Fc fragments were covalently linked via a PEG linker to the AFM tip and the mica sheet. A laser is directed to the cantilever tip and reflected onto a photodetector. Each interaction event leads to a deflection of the cantilever, detected by the laser reflected on the photodetector and a force-distance curve of a specific binding event was produced, together with a topography image and adhesion events. Quantification of DSG2 binding frequency expressed in relative interaction probability. B . in desmoplakin mutated AC patients (DPC), C . plakophilin 2 mutated patients (ARVC). D . Quantification of N-CAD binding frequency expressed in relative interaction probability in DPC. Each data point represents 1000 curves analyzed across two areas (10 µm x 10 µm). Data are presented as mean ± SEM.* p ≤ 0.05 compared to IgG, and NS is not significant compared to IgG. One-way ANOVA with Holm-Šídák’s multiple comparisons test was performed. N=3-5.

Article Snippet: As a positive control, two different anti-human DSG2 antibodies (DSG2-Origene, #BM5016; DSG2-Abcam, #ab14415) were used at 1:1000 dilutions in blocking buffer.

Techniques: Recombinant, Binding Assay, Produced