c-Src mediates α6β4 dependent mTOR activation. (A) MDA-MB-231breast cancer cells (parental, GFP and β4 shRNA infectants) and MDA-MB-435 human cancer cells (parental, mock and β4 shRNA infectants) were lysed using RIPA buffer. Equal amount of protein were isolated from extracts of these cell lined for Western blot analysis with indicated antibodies. (B) MDA-MB-231 cells and MDA-MB-435/ β4 cells were incubated with or without various concentration of PP2 for 24 hr before lysis by RIPA buffer. The laysate were analyzed by Western blotting using antibodies against phospho-mTOR (S2448), mTOR, phospho-Src (Y416), and Src. (C). MDA-MB-231 cells and MDA-MB-435/ β4 cells were infected with lentivirus that stably expresses either GFP or c-Src shRNA. These cells were lysed with RIPA buffer for Western blot analysis with indicated antibodies. The intensity of p-mTOR (S2448) 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.