Role of PRAS40 in H2O2-mediated reduction in 4E-BP1 phosphorylation. A549 cells were transfected with siRNA directed against PRAS40 (P) or scrambled (S) siRNA using the Amaxa system. Serum and leucine starved transfected cells were then treated with 250 μM H2O2 for 15 min prior to stimulating with leucine (0.8 mM) or insulin (174 nM) for an additional 30 min. Cells were then harvested, lysates separated by SDS-PAGE, and immunoblotted with antibodies directed against S6K1, PRAS40, 4E-BP1, S6K1 (Thr389); PRAS (Thr246), and non-phosphorylated (Np) 4E-BP1 (Thr46). Immunoblot is representative of 3 independent experiments.