FIG. 1.

Rapamycin inhibits phosphorylation of IRS-1 on Ser636/639 and enhances PI3-kinase/Akt signaling. (A) L6 myotubes were serum starved for 6 h, pretreated with rapamycin (Rap) for 30 min, and then treated with insulin for another 30 min as described in Materials and Methods, and endogenous IRS-1 was immunoprecipitated (IP). The presence of PI3-kinase in the immunoprecipitated material was determined by Western blotting (top right panel) and its activity was measured by TLC (left panel). The bottom right panel shows normalized IRS-1-associated PI3-kinase activity (mean values ± SEM) of the results from three independent experiments. (B and C) Total cell lysates treated as described for panel A were analyzed by Western blotting. (D) Normalized mean values ± SEM of results from three independent experiments shown in panel C; asterisks indicate that P values of <0.01. +, present; −, absent.