Effects of S279 and S282 mutation on Cx43-associated function and expression in neonatal ventricular myocytes. Cultured NRVMs were transduced with vector, wt-Cx43, S279A, S282A, or S282D mutant for 24 hours. S279/282 phosphorylation and Cx43 expression were detected by western blots (A). The statistical data (B) depict the effect of mutating S279 or S282 to alanine or aspartic acid on the relative abundances of pS279/282 and Cx43 expression. These transduced cells were also loaded with fluo-4 or LY dye, or immunostained with anti-Cx43 antibody, and then analyzed by confocal microscopy. Representative images (C) and the summarized data from 4–6 independent experiments illustrate the changes in coupled Ca2+ transients (D), LY uptake (E) and Cx43 distribution (bottom panels in C) because of Cx43 gene manipulation as indicated. Scale bar: 10 μm. **P?<?0.01 vs. vector cells; #P?<?0.05 and ##P?<?0.01 vs. wt-Cx43 cells in all panels, n = 4–8 independent determinations for each bar.