Fig. 8. KCl depolarization stimulates phosphorylation of ERKs 1/2 and CREB in rat myotubes in primary culture. (A) Top panel: representative western blots of ERKs of myotubes exposed to 84 mM KCl for the times indicated above the blots. Twenty µg of protein from whole cell lysates were analyzed by western blotting using an antibody that recognizes phosphorylated ERKs 1/2 (top blot). The blots were stripped and blotted with total ERKs antibody (bottom blot). Center panel: bars represent fold-induction of ERKs 1/2 phosphorylation (mean±s.e.m.) over control levels for three to six experiments. For A and B, *P<0.05, **P<0.001 (one-way analysis of variance followed by Dunnett’s multiple comparison post-test to compare the control to each of the conditions). Bottom panel: effect of 2-APB on ERKs phosphorylation. Myotubes were pretreated for 30 minutes with control vehicle or 50 µM 2-APB under resting conditions. Depolarization with 84 mM K+ was performed in the absence or presence of 2-APB and levels of P-ERKs were assayed at times shown. (B) Top panel: western blots of CREB phosphorylation following depolarization with high K+ solution for the times indicated. Fifty µg of protein from whole cell lysates were analyzed by western blotting with an antibody that recognizes CREB phosphorylated at serine 133. To correct for loading, a western blot with an antibody that recognizes the phosphorylated and nonphosphorylated forms of CREB was performed. Center panel: bars represent the fold induction (mean±s.e.m. for three to nine experiments) of CREB phosphorylation over control levels. Bottom panel: effect of 2-APB on CREB phosphorylation. Myotubes were pretreated for 30 minutes with control vehicle or 50 µM 2-APB under resting conditions. Depolarization with 84 mM K+ was performed in the absence or presence of 2-APB and levels of CREB and P-CREB were assayed at times shown.
