1. skMLCK physically interacts with and phosphorylates MEF2C in vivo and in vitro.
skMLCK physically interacts with and phosphorylates MEF2C in vivo and in vitro. (A) The in vivo interaction between MEF2C and skMLCK was observed in C2C12 myoblasts that were differentiated under serum starvation conditions. Co‐immunoprecipitation (IP) was performed using anti‐skMLCK antibodies conjugated to magnetic beads followed by western blot with antibodies against MEF2C. Anti‐IgG antibodies were used in a control IP. Intervening lanes have been removed for clarity, marked by a black line. (B) Flag‐tagged MEF2C and His‐tagged skMLCK or its mutants were co‐transfected into HEK‐293 cells. Co‐immunoprecipitation (IP) using anti‐Flag‐agarose resin was followed by western blot analysis (WB) with anti‐His antibodies. WB for His‐, Flag‐, and β‐actin show expression prior to IP. Intervening lanes have been removed for clarity, marked by a black line. (C) In vitro kinase assays were performed with recombinant His‐MEF2C incubated with purified skMLCK as indicated and visualized by silver stain or autoradiography. (D) In vivo kinase assays were performed in HEK‐293 cells co‐transfected as indicated. After immunopurification with an anti‐Flag resin, western blot analysis and autoradiography were performed. An Image J program was used to measure band intensities and the intensity of each 32P‐radiolabelled band was normalized to the corresponding level of MEF2C protein. The data are shown as the normalized average 32P intensity±s.e.m. (n=3). (E) The MEF‐domain protein sequence alignment from different species, showing conservation of T80. *P<0.05.
Ashraf Said Al Madhoun et al. EMBO J. 2011;30:
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