Fig. 1. Phosphorylation of various TORC1 components in response to EtOH.
C2C12 myocytes were incubated in the presence or absence of 100 mM EtOH for 18-24 h. Equal amounts of cell extracts were collected and analyzed via Western blotting using antibodies against phosphorylated (P) mTOR (S2448), raptor (S792), PRAS40 (T246), and Akt (T308, S473) as well as the total (T) forms of the indicated proteins (panel A). Panel B, phosphorylation of mTOR, raptor, and PRAS40 was quantified from 5 independent experiments and plotted on a graph (3 replicate samples/experiment). Results for indicated phosphorylated proteins were normalized to total protein and expressed as a percentage of basal control levels. Data are mean ± SE. * P< 0.05 versus control (con) values. Panel C, myocytes were incubated with insulin (20 nM) for 15 min or with AICAR (2 mM) for 2.5 h. Cell lysates were analyzed by immunoblotting with total and phosphorylated PRAS40 (T246).