Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: (A) Clear expression of mRNA can be detected for most desmogleins, with the exception of DSG1 which shows only faint expression. ( B–C ) Representative immunofluorescence analyses of DSG2 (B) and DSG4 (C) at the cell border of normal human prostatic glandular epithelium. Original magnification: 200X. Scale bars correspond to 100 µm.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques: Expressing, Immunofluorescence

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: (A–C) DSG2 is expressed in the luminal cells of the prostatic glands and this expression rarely co-localizes with basal cell CK14 expression. ( D–F ) DSG2 co-localizes with PSA expression in the luminal compartment of the gland. ( G–I ) DSG4 is also expressed in the luminal cells and rarely co-localizes with basal cell CK14 expression. ( J–L ) DSG4 expression co-localizes with luminal cell PSA expression. ( M–O ) The expression of DSG4 also shows almost complete co-localization with that of DSG2 in the luminal cells. Original magnification: 200X. Scale bars correspond to 100 µm.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: (A) DSG2 expression is detected in all cell lines examined, including the normal immortalized prostate cell line BPH-1. Data is represented as mean ± SD. **P<0.01; *** P<0.001. ( B–E ) DSG2 is expressed at the cell border of LNCaP and DU145 cells; however, cell border expression is not detected in PC3 cells with only some cells showing faint, punctate, and diffuse staining (insert magnification; shown at 2.5X). Original magnification: 200X. Scale bars correspond to 100 µm.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: Expression of DSG2 in prostate cancer as compared to normal prostate.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques: Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: (A–L) Representative immunofluorescence analysis of DSG2 and CK8/18 in prostate cancer. ( D, E–H ) TMAs showing high DSG2 expression in the cell border of well differentiated areas of the tumor (panel D, inside yellow dashed line). ( D, I–L ) TMAs showing low DSG2 expression in poorly differentiated areas of the tumor (panel D, shown in the remainder of the tumor outside of the yellow dashed line). Original magnification: 200X. Scale bars correspond to 100 µm. ( M ) Patients expressing higher levels of DSG2 had a significantly longer recurrence free survival than those expressing lower levels of DSG2 ( P = 0.01).

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques: Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: Correlation between DSG2 and clinico-pathological characteristics.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques:

Journal: PLoS ONE

Article Title: Characterization of Desmoglein Expression in the Normal Prostatic Gland. Desmoglein 2 Is an Independent Prognostic Factor for Aggressive Prostate Cancer

doi: 10.1371/journal.pone.0098786

Figure Lengend Snippet: Multivariate analyses in the 414 patient cohort.

Article Snippet: Anti-DSG2 mouse monoclonal antibody (clone 10G11) was purchased from ARP, Inc (Belmont, MA) and used at a dilution of 1∶50 for immunofluorescence analysis of normal prostate tissue and cell lines; anti-DSG1/2 mouse monoclonal antibody (clone DG3.10) was purchased from Fitzgerald (Acton, MA) and used at a dilution of 1∶20 for TMAs.

Techniques:

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: Scientific Reports

Article Title: Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase

doi: 10.1038/s41598-017-06713-y

Figure Lengend Snippet: Inhibition of Dsg2 binding resulted in increased p38MAPK activity which is critical for enterocyte cohesion. ( A ) Western blot analysis after incubation of DLD1 cells with a Dsg2-specific antibody for 30 min revealed increased phosphorylation of p38MAPK. Cropped blots are displayed and full-length blots are included in the supplementary information. ( B ) Band intensity of detected p-p38MAPK was quantified using ImageJ and normalized to control (shown is mean ± SE, n = 6, *p < 0,05 compared to control). ( C ) DLD1 cells were treated with the p38MAPK inhibitor SB202190 or the activator anisomycin and analysed in a dispase-based cell dissociation assay. Both resulted in increased cell monolayer fragmentation. (Shown is mean ± SE, n = 3, *p < 0,05 compared to control).

Article Snippet: Following primary antibodies were used: mouse anti Dsg2 (clone 10G11) and rabbit anti Dsg2 (rb5, both Progen, Heidelberg, Germany), rabbit anti DP (NW6, USA, self-made), mouse anti Dsc2/3 (clone 7G6, Life Technologies, Carlsbad, CA), rabbit anti Claudin-4, rabbit anti Claudin-1 and rabbit anti Claudin-2 (all from Life Technologies, Carlsbad, CA), mouse anti E-cadherin (clone 36, BD Bioscience, Heidelberg, Germany,) mouse anti GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti α-tubulin (Abcam, Cambridge, UK), rabbit anti p38MAPK, rabbit anti phospho-Thr180/182 p38MAPK, (Cell Signaling, Danvers, MA, USA),.

Techniques: Inhibition, Binding Assay, Activity Assay, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase

doi: 10.1038/s41598-017-06713-y

Figure Lengend Snippet: Well-balanced p38MAPK activity is critical for intestinal barrier function. Ca 2+ -switch assay with confluent DLD1 cells. ( A ) TER values decrease during depletion with 4 mM EGTA for 1 h and increase to back to control values during 2 h of repletion with 8 mM CaCl 2 . ( B ) Immunostaining of Dsg2 and Cld4 shows reduced and fragmented staining as well as gaps after 1 h depletion and similar staining to control condition after 2 h repletion. ( C ) Barrier reformation is not disturbed after application of a Dsg2-specific antibody but is impaired after addition of an inhibitory anti E-cadherin antibody. ( D ) TER values decrease after inhibition of p38MAPK with SB202190 and also barrier reformation is impaired in the Ca 2+ -switch experiment. Activation of p38MAPK via anisomycin has no effect in the short run but also induces reduction of TER values after about 10 h. (shown are representative graphs for at least three independent experiments).

Article Snippet: Following primary antibodies were used: mouse anti Dsg2 (clone 10G11) and rabbit anti Dsg2 (rb5, both Progen, Heidelberg, Germany), rabbit anti DP (NW6, USA, self-made), mouse anti Dsc2/3 (clone 7G6, Life Technologies, Carlsbad, CA), rabbit anti Claudin-4, rabbit anti Claudin-1 and rabbit anti Claudin-2 (all from Life Technologies, Carlsbad, CA), mouse anti E-cadherin (clone 36, BD Bioscience, Heidelberg, Germany,) mouse anti GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti α-tubulin (Abcam, Cambridge, UK), rabbit anti p38MAPK, rabbit anti phospho-Thr180/182 p38MAPK, (Cell Signaling, Danvers, MA, USA),.

Techniques: Activity Assay, Control, Immunostaining, Staining, Inhibition, Activation Assay

Journal: Scientific Reports

Article Title: Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase

doi: 10.1038/s41598-017-06713-y

Figure Lengend Snippet: Dsg2 regulates p38MAPK activity which is required for barrier properties. ( A ) Comparison of baseline TER values between wildtype and knockout cells revealed reduced TER in DLD1 cells lacking Dsg2 (shown is mean ± SE, n ≥ 10, *p < 0,05 compared to control, n.s not significant). ( B ) During Ca 2+ depletion for 1 h TER values of Dsg2-deficient cells decreased stronger compared to wildtype cells. (Shown is mean ± SE, n ≥ 6, *p < 0,05 compared to control, n.s not significant). ( C ) Wildtype and knockout cells grown on coverslips were stained for Dsg2 and Dsc2 to confirm knockout. Scale bar, 10 µm ( D ). Level of p-p38MAPK in Dsg2 and Dsc2 knockout cells was analysed via Western blot. Loss of Dsg2 and Dsc2 resulted in reduced level of p-p38MAPK which was not restored by Dsc2 rescue. α-Tubulin served as loading control. Cropped blots are displayed and full-length blots are included in the supplementary information. ( E ) Band intensity of detected p-p38MAPK was quantified using ImageJ and normalized to control (shown is mean ± SE, n ≥ 3, *p < 0,05 compared to control, n.s not significant). ( F ) Time for complete repletion during Ca 2+ -switch experiments was compared between wildtype and double knockout of Dsg2 and Dsc2. Loss of desmosomal cadherins resulted in a prolonged repletion time which was rescued by activation of p38MAPK via anisomycin (shown is mean ± SE, n ≥ 6, *p < 0,05 compared to wildtype under control conditions, #p < 0,05 compared to ΔDsg2ΔDsc2 under control conditions, n.s not significant). ( G ) Treatment with anisomycin reduced repletion time in ΔDsg2ΔDsc2 knockout cells. Representative graph for at least four independent Ca 2+ -switch experiments is shown.

Article Snippet: Following primary antibodies were used: mouse anti Dsg2 (clone 10G11) and rabbit anti Dsg2 (rb5, both Progen, Heidelberg, Germany), rabbit anti DP (NW6, USA, self-made), mouse anti Dsc2/3 (clone 7G6, Life Technologies, Carlsbad, CA), rabbit anti Claudin-4, rabbit anti Claudin-1 and rabbit anti Claudin-2 (all from Life Technologies, Carlsbad, CA), mouse anti E-cadherin (clone 36, BD Bioscience, Heidelberg, Germany,) mouse anti GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti α-tubulin (Abcam, Cambridge, UK), rabbit anti p38MAPK, rabbit anti phospho-Thr180/182 p38MAPK, (Cell Signaling, Danvers, MA, USA),.

Techniques: Activity Assay, Comparison, Knock-Out, Control, Staining, Western Blot, Double Knockout, Activation Assay

Journal: Molecular Medicine Reports

Article Title: Desmocollin-2 affects the adhesive strength and cytoskeletal arrangement in esophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2014.2485

Figure Lengend Snippet: RNA interference-mediated inhibition of DSC2 affects desmosome protein expression and localization. (A) Immunofluorescence analysis of the subcellular localizations of the DSG2 and PKP2 proteins (magnification, ×400). Of note, knocking down the expression of DSC2 caused reduced DSG2 and PKP2 membrane localization (white arrows). (B) Western blot analyses of DSG2 and PKP2 protein expression in the DSC2-specific siRNA or control-transfected SHEEC cells. β-actin served as a loading control. DSC2, desmocollin-2; siDSC2, DSC2-specific siRNA; DSG2, desmoglein-2; PKP2, plakophilin-2.

Article Snippet: The following antibodies were used: Mouse monoclonal anti-DSC2 (7G6; Invitrogen Life Technologies), mouse monoclonal anti-DSG2 (10G11; Progen Biotechnik GmbH, Heidelberg, Germany), mouse monoclonal anti-PKP2 (PP2/62, PP2/86, PP2/150; Progen Biotechnik GmbH) and mouse monoclonal anti-β-actin (Sigma, St. Louis, MO, USA).

Techniques: Inhibition, Expressing, Immunofluorescence, Western Blot, Transfection