Journal: PLoS ONE

Article Title: In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: Investigated DSG2-variants.

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (anti-DSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: Schematic view of the rECD with analysed ARVC-associated variations. The dotted line shows the predicted PC cleavage site. SS = signal sequence, Pro = prodomain, EC1-EC4 = DSG2 extracellular cadherin subdomains 1-4. B Recombinantly expressed proteins were identified as DSG2-ECD with anti-DSG2-10G11 by Western blot analysis. The calculated apparent molecular weights were 67.5±1.5, 72.5±3.5, 70.0±3.0, 70.0±3.0, 70.5±2.5, and 69.0±4.0 (mean±SEM; n = 2) for the proteins in the traces in 1, 2, 3, 4, 5 and 6, respectively. C Coomassie-R-250 staining revealed the purity of the proteins. 1 = rECD-wt, 2-6 = rECDs as labelled in A .

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (anti-DSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Sequencing, Western Blot, Staining

Journal: PLoS ONE

Article Title: In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: A+B Flow cytometry-based assay for the binding of 0.8 µM rECD-wt or -variants to HT1080. A Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti-HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). B Column plots representing the ratio of rECD-binding related to the negative control (ratio rECD-bound ) as detected by flow cytometry. Ratios rECD-bound are indicated as mean± SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. C Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl 2 containing buffer with BS 3 (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligomers (o).

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (anti-DSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Flow Cytometry, Binding Assay, Fluorescence, Negative Control, Incubation, Western Blot

Journal: PLoS ONE

Article Title: In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: DSC2b-HT1080 cells were transfected with full-length(fl)-DSG2-pEYFP; live cells were analysed with a fluorescence miscroscope one day after transfection. R46Q, D154E, D187G, K294E and V392I indicate the sequence variant in fl-DSG2-EYFP, wt fl-DSG2-wt-pEYFP, and C the LFA mock transfected control. Chimeric DSG2-proteins localised preferentially to the cell borders. ARVC-associated variations had no detectable influence on the localisation of fl-DSG2-EYFP in DSC2b-HT1080. Images were acquired through YFP and phase-contrast filters. Scale (red bar) = 10 µm.

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (anti-DSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Transfection, Fluorescence, Sequencing, Variant Assay

Journal: Frontiers in Immunology

Article Title: Role of Dsg1- and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion

doi: 10.3389/fimmu.2019.01128

Figure Lengend Snippet: Genetic deletion of Dsg3 and Dsg2 using CRISPR/Cas9 in HaCaT keratinocytes. (A) Schematic of sequencing results after inducing a DSB with NHEJ repair in Exon 5 of either Dsg3 or Dsg2 using CRISPR/Cas9. (B) Cell line characterization by immunostaining of desmosomal proteins ( n = 3). (C) Cell line characterization by immunoblot with representative images on the left and densitometric quantification on the right ( n = 3, one-way ANOVA, * p ≤ 0.05 vs. WT). (D) Dispase assay of WT and Dsg-deficient cell lines under baseline treatment. ( n = 7, one-way ANOVA, * p ≤ 0.05 vs. WT) (E) Cell lines incubated with IgG fractions for 24 h and subjected to Dispase assays ( n = 5, two-way ANOVA, # p ≤ 0.05 vs. respective c-IgG, * p ≤ 0.05 vs. WT).

Article Snippet: Membranes were blocked by 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween (TBS-T) for 1 h at room temperature and following primary antibodies were used overnight at 4°C in blocking solution: phospho-EGF Receptor mAb (Tyr845) (Cell Signaling Technologies, Danvers, USA), EGF Receptor mAb (CST), phospho-p44/42 MAPK mAb (CST), p44/42 MAPK mAb (CST), GAPDH (Santa Cruz, Heidelberg, Germany), Desmoplakin I/II (H-300) (Santa Cruz), α-Tubulin (Abcam, Cambridge, UK), Dsg3 pAb (Biozol, Eching, Germany), PG (Progen, Heidelberg, Germany), E-Cad (BD Transduction), Dsg2 (OriGene, Herford, Germany), Desmocollin (Dsc) 3 (Progen).

Techniques: CRISPR, Sequencing, Immunostaining, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Role of Dsg1- and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion

doi: 10.3389/fimmu.2019.01128

Figure Lengend Snippet: Signaling pathway modulation by pemphigus IgG fractions in Dsg-deficient cell lines. Analysis of ERK activation after 30 min application of IgG fractions by immunoblot in Dsg3- and Dsg2-deficient cell lines. (A) Representative immunoblot and (B) densitometric analysis ( n = 9, two-way ANOVA, * p ≤ 0.05). (C) Representativ graph of Fura-2 ratiometric Ca 2+ imaging reveals a PF-IgG mediated Ca 2+ influx in all cell lines used ( n = 3).

Article Snippet: Membranes were blocked by 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween (TBS-T) for 1 h at room temperature and following primary antibodies were used overnight at 4°C in blocking solution: phospho-EGF Receptor mAb (Tyr845) (Cell Signaling Technologies, Danvers, USA), EGF Receptor mAb (CST), phospho-p44/42 MAPK mAb (CST), p44/42 MAPK mAb (CST), GAPDH (Santa Cruz, Heidelberg, Germany), Desmoplakin I/II (H-300) (Santa Cruz), α-Tubulin (Abcam, Cambridge, UK), Dsg3 pAb (Biozol, Eching, Germany), PG (Progen, Heidelberg, Germany), E-Cad (BD Transduction), Dsg2 (OriGene, Herford, Germany), Desmocollin (Dsc) 3 (Progen).

Techniques: Activation Assay, Western Blot, Imaging