Effect of S282 mutation on Cx43 expression in endogenous Cx43-knockdown neonatal ventricular myocytes. NRVMs were transduced with scramble or siRNA to knockdown Cx43 (Cx43-KD, m.o.i. = 60) for 48 hours followed by further exposure of wt-Cx43 (m.o.i = 20) or S282A mutant (m.o.i. = 2) for 24 hours. The pS279/282 levels in triton X-100 soluble and insoluble fractions was determined by western blot (A), and normalized by the level of actin and then the level of Cx43 in scramble cells (B). The detection of GAPDH observed in non-junctional but not in junctional fraction represents a successful separation of non-junctional and junctional Cx43. N = 4 independent determinations for each bar, and ** denotes P?<?0.01 vs. scramble cells. Cultured NRVMs were co-immunostained with anti-Cx43 and anti-HA-probe antibodies after transduced with scramble, wt-Cx43 (m.o.i = 20), S282A-HA (m.o.i = 2), or S282D-HA mutant (m.o.i = 2) for 24 hours (C). Representative confocal images show the subcellular distributions of total Cx43 (green), exogenous Cx43-HA (red) and their co-localizations in S282D-HA and S282A-HA cells (yellow). Scale bar: 10 μm.