Anti cx50 rabbit polyclonal antibody

Cells at about 90% confluence were collected in ice-cold phosphate buffered saline (PBS) and centrifuged at 1000 rpm at 4 °C for 5 min. Total protein was extracted from the stably transfected Hek293 cells by means of lysis buffer supplemented with protease inhibitors (Sangon Biotech, Shanghai, China). The protein concentration was estimated using a bicinchoninic acid (BCA) assay kit (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, followed by resolving using 10% SDS-polyacrylamide gel electrophoresis and transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The extracts were subjected then to immunoblotting with a 1:1000 dilution anti-Cx50 rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), as well as a 1:5000 dilution anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C, overnight. Fluorescent secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:5000 dilution was used for incubation. The blots were analyzed using a ChemiDoc^TM MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA), in which the expression levels of the target protein were normalized relative to β-actin expression.

The expression levels of Cx50WILD and Cx50V44A in stably transfected HeLa cells were verified by Western blot, and the EGFP-transfected cells served as controls. The target protein was blotted with anti-Cx50 rabbit polyclonal antibody (1∶200 dilution, Santa Cruz) and with fluorescent secondary antibodies, and its expression level was normalized relative to GAPDH expression.