Cells cultured on 100-mm dishes were rinsed twice with PBS and harvested in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease/phosphatase inhibitors. After protein concentration measurement, equal amounts of each sample were mixed with sample buffer, denatured by heating at 95°C for 10 minutes, and subjected to electrophoresis on 4% to 20% Tris-Glycine gels (Invitrogen, Grand Island, NY). The protein bands were transferred to nitrocellulose membranes and equal loading was verified by Ponceau S staining. The membranes were incubated in 5% BSA in Tris-buffered saline (TBS) for 1 hour followed by an overnight incubation (4°C) with primary antibodies at the optimal concentration. The membranes were washed with TBS with 0.03% Tween 20 and incubated with the horseradish peroxidase (HRP)–conjugated secondary antibody for 1 hour at room temperature. Detection was performed with ECL Plus Western Blotting Detection System (Amersham, Buckinghamshire, UK) and recorded on film (HyBlot CL; Denville Scientific, Inc., Metuchen, NJ). The following antibodies were used: rabbit anti–phospho-mTOR (Ser2448), rabbit anti–phospho-S6 ribosomal protein (Ser240/244, 61H9), rabbit anti-p70 S6 kinase, rabbit anti–phospho-p70 S6 kinase (Thr389, 108D2), and rabbit anti-GAPDH, all from Cell Signaling Technology, Inc. (Danvers, MA). Also used were rabbit antifibronectin (H-300) and mouse anti–α-actin (1A4), from Santa Cruz Biotechnology (Santa Cruz, CA).