c-Src is required for α6β4 dependent TORC1 and TORC2 activation. (A) MDA-MB-231 cells and MDA-MB-435/ β4 cells were incubated with DMSO and indicated concentrations of PP2 for 24 hr before lysis by RIPA buffer. Equal amount of protein were isolated and analyzed by Western blotting using antibodies against phospho-AKT (S473), AKT, phospho-S6 ribosomal protein (S235/236), and phospho-4E-BP1 (S65). (B) Equal lysates from MDA-MB-231 cells and MDA-MB-435/ β4 cells expressing shRNA against GFP or c-Src were analyzed by Western blotting with indicated antibodies. The intensity of p-4E-BP1 (S65) band was quantified by densitometric analysis using ImageJ software. The number given underneath gel image represents fold change compared with control cells. All results are representative of three independent experiments.