Western Blot Analysis of mTOR Pathway

Western blotting was performed as previously described [41 (link)]. In brief, cell lysates were prepared in NP40 buffer and separated using SDS gel electrophoresis. Afterwards, the proteins were transferred onto a nitrocellulose membrane and incubated with specific primary antibodies against mTOR (#2983, Cell Signaling Technology, Danvers, MA, USA), panAKT (#4685, Cell Signaling Technology), pAKT S473 (#4060, Cell Signaling Technology), S6 (#2217, Cell Signaling Technology), pS6 S240/S244 (#5364, Cell Signaling Technology), pGSK3ß S9 (#9336, Cell Signaling Technology) or HSC70 (#sc-7298, Santa Cruz Biotechnology, Dallas, TX, USA). Afterwards, the membrane was incubated with the appropriate secondary antibodies against anti-mouse-IgG (#7076, Cell Signaling Technology) or anti-rabbit-IgG (#7074, Cell Signaling Technology). The protein expression was analyzed using the LAS-3000 or LAS-4000 Imager from Fuji (Raytest, Straubenhardt, Germany). Densitometric quantification was carried out using AIDA Image Analyser Software Version 3 (Elysia-raytest GmbH, Straubenhardt, Germany).