Fig. 4

p38α mediates phosphorylation of KMT1A during myoblasts differentiation. a Western blot analysis of cell extracts from C2-4RE-luc/KMT1A-F expressing control vector (Em), MKK6EE or MKK6DN via lentiviral delivery grown in GM, and C2C12 grown in DM as positive control of differentiation, probed with antibodies for indicated proteins, and β-actin as for loading control. Phosphorylated KMT1A was detected C2-4RE-luc cells expressing only MKK6EE in GM and C2C12 cells in DM. b Western blot analysis of cell extracts from C2C12 grown in GM or DM at various time points, probed with antibodies for detecting indicated proteins, GAPDH as loading control. An increased level of phosphorylated KMT1A was observed with increased activated p38 status during the differentiation of C2C12 myoblasts. c Western blot analysis of cell extracts from C2C12 grown in GM or DM in the presence or absence of SB, probed with antibodies against indicated proteins. GAPDH served as loading control. Inhibition of KMT1A phosphorylation was observed by SB-treatment-induced blockade of p38a activation in cells under DM conditions. d Western blot analysis of cell extracts from C2C12 cells expressing scramble control shRNA (Ctrl) or p38αshRNA via lentiviral delivery grown as specified in GM or DM, probed with antibodies for indicated proteins, where GAPDH for loading control. Inhibition of KMT1A phosphorylation was observed by p38α knockdown. e Control IgG or anti-Flag (M2) immunoprecipitates were retrieved as indicated from extracts of 293A cells expressing with or without Flag-KMT1A via lentiviral delivery. These immunoprecipitates were then subjected to in vitro kinase assay in the presence of purified p38α with or without SB. Subsequently, the reaction mixture was divided equally and evaluated one part for autoradiography and another part for western blot analysis. Phosphorylation of KMT1A directly by p38α was observed