Fig. 4. p38-MAPK and PI3K are activated in proliferation and are not controlled by the differentiation-linked hyperpolarization. (A) Activation of p38-MAPK pathway was detected by western blot using an antibody directed against phospho-p38-MAPK (Thr180/Tyr182). Phospho-p38-MAPK is present in proliferating myoblasts (GM), is maintained in differentiating myoblasts (DM) and depolarization induced by 10 mM Cs⁺ (Cs10 mM) does not affect the level of phosphorylation. By contrast, myogenin expression associated with differentiation is reduced in presence of Cs⁺. Protein extracts were prepared at the indicated time. (B) Phosphorylation of recombinant ATF2 by immunoprecipitated phospho-p38-MAPK was used to assess p38-MAPK activity. ATF2 phosphorylation has been detected with an antibody specific for phospho-ATF2 (Thr71). Phospho-p38-MAPK was immunoprecipitated from proliferating myoblasts (GM), and from myoblasts differentiated for 4 and 24 hours in presence or absence of 10 mM Cs⁺. (C) Detection of phospho-AKT by western blot. Myoblasts were grown in media without exogenous insulin. Phospho-AKT (Ser473/Thr308) is present in proliferating myoblasts (GM) and myoblasts differentiated for 24 hours (DM). Phospho-AKT expression is not affected by 10 mM Cs⁺ (Cs10) or 15 µM BAPTA-AM (BA). Myogenic differentiation is confirmed by myogenin expression.