human desmoglein 2 fc chimera protein  (R&D Systems)

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: First, Nunc MaxiSorpTM flat-bottom plates (Thermo Scientific; # 44-2404-21) were coated with 2 μg ml -1 recombinant human DSG2 (Creative Biomart; #DSG2-1601H) in 100 mM bicarbonate/carbonate coating buffer (3.03 g Na2CO3; 6.0 g NaHCO3; ad.

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: First, Nunc MaxiSorpTM flat-bottom plates (Thermo Scientific; # 44-2404-21) were coated with 2 μg ml -1 recombinant human DSG2 (Creative Biomart; #DSG2-1601H) in 100 mM bicarbonate/carbonate coating buffer (3.03 g Na2CO3; 6.0 g NaHCO3; ad.

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: First, Nunc MaxiSorpTM flat-bottom plates (Thermo Scientific; # 44-2404-21) were coated with 2 μg ml -1 recombinant human DSG2 (Creative Biomart; #DSG2-1601H) in 100 mM bicarbonate/carbonate coating buffer (3.03 g Na2CO3; 6.0 g NaHCO3; ad.

Techniques: Recombinant, Binding Assay, Produced