anti-human dsg2 antibody dsg2-origene  (OriGene)

Journal: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-04853-1

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 three 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: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-04853-1

Figure Lengend Snippet: In vitro DSG2 and N-CAD cleavage assays. A Human DSG2-Fc protein (200 ng/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 h, without (C) and with protease inhibitor (I, cOmplete™), and Western blot analysis was performed. A representative image of three 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: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-04853-1

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 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 × 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

Journal: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-04853-1

Figure Lengend Snippet: Catalytic antibodies mediated cardiomyocyte cohesion in AC patients. In normal healthy individuals, the cadherin family proteins, desmoglein 2 (DSG2) and N-cadherin (N-CAD) proteins are properly localized to the ICD via the plaque proteins DSP and PKP2 and armadillo proteins PG, α-catenin (α), β-catenin (β) and YAP (Y), respectively. In AC patients, having mutations in either DSP or PKP2 genes (the corresponding proteins are represented with dashed boxes), presence of catalytic antibodies (CA) that can cleave DSG2 or N-CAD could destabilize the cadherin-mediated adhesion (1), which could lead to the activation of p38MAPK (2), which further can lead to desmosome destabilization. Cleavage of cadherin proteins combined with p38MAPK activation could eventually result in reduced cardiomyocyte cohesion (3) and thereby a reduction in the mechanical strengths of the cardiomyocyte. Cartoon created with BioRender.com

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: Activation Assay

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: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: Immunofluorescence, Incubation, 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: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: In Vitro, Incubation, 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: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: Recombinant, Binding Assay, Produced