Figure 4.

Insulin stimulation in L6GLUT4myc muscle cells causes dephosphorylation of cofilin that is dependent on SSH1. L6GLUT4myc myoblasts were serum-starved for 3 h before insulin stimulation (100 nM) for the indicated time periods. (A) Total lysates immunoblotted for P-cofilin, cofilin, P-Akt, and actin (as loading control). Representative blots of four experiments are shown. (B) Quantification of insulin-dependent cofilin dephosphorylation expressed as P-Cofilin/Cofilin ratio relative to time 0 (n = 4, mean ± SE, ^#p < 0.05). (C) Lysates from myoblasts treated with SSH1 siRNA (siSSH1) were prepared to determine the knockdown effect on SSH1 and its contribution toward insulin-induced cofilin dephosphorylation and Akt phosphorylation by immunoblotting for P-cofilin and p-Akt (Ser-473). Myoblasts were (D) transfected with p34 siRNA or (E) subjected to 250 nM LB treatment for 30 min, and the effect of insulin on P-Cofilin was assessed. Representative blots of more than three independent experiments are shown.