Anti cx43 rabbit polyclonal antibody

Cx50WILD-transfected, Cx50V44A-transfected, EGFP-transfected HeLa cells and human retinal epithelial cells (ARPE-19, ATCC) were seeded in 100-mm dishes for 24 h. ARPE-19, which has been reported to express Cx43 [10] (link), served as a control. The cells were collected in ice-cold PBS and were then centrifuged at 1000 rpm at 4°C for 5 min. After removing the supernatant, total protein was extracted from the cells with a lysis buffer (Sangon Biotech, Shanghai, China). After incubation on ice for 30 min, the extracts were centrifuged at 14,000 rpm at 4°C for 15 min. Then, the supernatant of each sample was transferred to a fresh tube and boiled with a protein-loading buffer (Sangon Biotech). The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes and blotted with anti-Cx43 rabbit polyclonal antibody (1∶200 dilution, Santa Cruz) and anti-GAPDH rabbit monoclonal antibody (1∶5000 dilution, Cell Signaling Technology, USA). After incubation in fluorescent secondary antibodies (1∶5000 dilution, Cell Signaling), the blots were analyzed using the Bio-Rad ChemiDoc MP imaging system. The target protein expression levels were normalized relative to GAPDH expression.

HLECs transiently transfected with plasmids or stable HEK-293 cells lines were harvested, and total protein was extracted using a RIPA protein extraction kit (Beyotime, China). Proteins were separated by 12% SDS-polyacrylamide gels and individually transferred to methanol-activated polyvinylidene difluoride membranes (Millipore, USA). The membranes were immunoblotted with a 1:1000 dilution of anti-Flag mouse monoclonal antibody (Sigma, CAS No. f1804, USA) or a 1:200 dilution of anti-Cx43 rabbit polyclonal antibody (Santa Cruz, CAS No. sc-9059, USA). A 1:1000 dilution of anti-GAPDH rabbit antibody (Diagbio, CAS No.db106, China) was used as an internal control. The fluorescent secondary goat anti-mouse (LI-COR Biosciences, CAS No.926-68072, USA) or goat anti-rabbit (LI-COR Biosciences, CAS No.925-32211, USA) antibodies (1:5000) were used for visualization. The signals were analyzed using the Image J program. The target protein expression levels were normalized relative to GAPDH expression. Each experiment was repeated at least three times.