Western blot analysis

Proteins were extracted from tumors in RIPA buffer (50 mM Tris HCL pH 8, 150 mM NaCl, 0.5% deoxycholic acid, 0.5% Triton), supplemented with protease and phosphatase inhibitors. Lysates were resolved by electrophoresis in 10% agarose gels. The resulting bands were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then probed with rabbit antibodies against AKT, p-AKT (Ser473), S6, p-S6, 4E-BP1, p-4E-BP1, PRAS, p-PRAS and GAPDH (Cell Signaling®). The membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated affinity-purified goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Interchim). Protein was quantified with Multi Gauge software and normalized against GAPDH levels.