Mouse monoclonal anti dsg2 10g11

Staining was performed, as previously described (6 (link)). The cells were fixed in 4% paraformaldehyde solution (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 15 min, following which they were incubated with donkey serum blocking buffer [Tris-buffered saline containing 5% (w/v) normal donkey serum (Jackson ImmunoResearch, Hamburg, Germany)] for 30 min and the primary antibody for 60 min. Subsequently, the cells were incubated with donkey anti-mouse polyclonal immunoglobulin G (IgG) and/or donkey anti-rabbit polyclonal IgG (DyLight 488 and 594, respectively; Jackson ImmunoResearch) for 60 min at room temperature. The following antibodies were used: Mouse monoclonal anti-DSC2 (7G6; Invitrogen Life Technologies), rabbit polyclonal anti-E-cadherin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-γ-catenin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-DSG2 (10G11; Progen Biotechnik GmbH), mouse monoclonal anti-PKP2 (PP2/62, PP2/86, PP2/150, Progen Biotechnik GmbH), mouse monoclonal anti-pan-cytokeratin (Santa Cruz Biotechnology, Inc.) and Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton Inc., Denver, CO, USA).

Total cell lysates were prepared in radioimmunoprecipitation assay buffer, separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated in 10 ml of blocking buffer [Tris-buffered saline containing 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dried milk powder (Sangon Biotech, Shanghai, China)] for 1 h at room temperature and then incubated with the indicated antibody. Finally, immunoreactive bands were revealed using a luminol reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Images were captured and quantitative analyses were performed using FluorChem™ IS-8900 (Alpha Innotech Co., San Leandro, CA, USA). 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).