Detailed Western Blot Protocol

Western blots were performed as previously described.^{13 (link)} Briefly, sample were subjected to separation on 10% SDS–PAGE gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked in 5% non-fat dry milk, and were incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Membranes were visualized using Clarity ECL western blotting substrate (Bio-Rad, Hercules, CA, USA), and imaged with a UVP EpiChem gel documentation system (UVP Bioimaging Systems, Upland, CA, USA). The rabbit anti-Cx43 antibody was purchased from Sigma. The mouse anti-GAPDH and rabbit anti-osterix antibodies were purchased from Millipore.