P s6 ribosomal protein s6k ser235 ser236

Immunoblotting was performed as described previously [18 (link)]. The following antibodies were used: α-tubulin and β-actin (Sigma-Aldrich), p21^Cip1/WAF1 and p27^KIP1 (BD-Pharmingen, San Diego, CA); p53 (DO-7; Dako, Carpinteria, CA); BRCA1(Santa Cruz Biotechnology, Dallas, TX); Cdc2 (MBL, Nagoya, Japan); PIM-1 and XPO1 (marketed as anti-CRM1) and p-HSF1^Ser326 (Abcam, Cambridge, MA); CDC25C, c-Myc, cyclin D1, Hsp70, 4E-BP1, phosphorylated- (p-) 4E-BP1^Thr37/Thr46, p-Rb^Ser780, S6 (S6K), p-S6 ribosomal protein (S6K)^{Ser235/Ser236}, PUMA, HSF1, Hsp70 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG (all from Cell Signaling Technology).

Cells were solubilized in lysis buffer comprising phosphate-buffered saline solution containing 1× cell lysis buffer (Cell Signaling Technology, Danvers, MA), 1× protease inhibitor cocktail (Roche, Indianapolis, IN), and 1× phosphatase inhibitor cocktail (Roche) and incubated for 30 min on ice. The lysates were then subjected to centrifugation for 10 min at 13,000 rpm at 4°C. Total protein concentrations were determined by the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Total proteins (40 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad Laboratories) and transferred to polyvinylidene-fluoride membranes (0.45 μm, Millipore, Bedford, MA), then probed with first and second antibodies according to the manufacturers’ protocols. The following antibodies were used: α-tubulin (Sigma-Aldrich, St Louis, MO), p-HSF1^Ser326 (Abcam, Cambridge, MA), HSF1, p-S6 ribosomal protein (S6K) ^{Ser235/Ser236}, S6 ribosomal protein, c-Myc, cleaved caspase-9, p-AMPKa^Thr172, AMPKα, p-TSC2^Thr1462, TSC2 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG (all, Cell Signaling Technology).

Immunoblot analysis was performed as previously described^{38 (link)}. The following antibodies were used: α-tubulin (Sigma-Aldrich, St Louis, MO), LC-3 (MBL, Nagoya, Japan), phosphorylated- (p-)4E-BP1 Thr37/Thr46, 4E-BP1, p-S6 ribosomal protein (S6K) Ser235/Ser236, S6 ribosomal protein, p-AMPKα Thr172, AMPKα, ATF4 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG (all, Cell Signaling Technology, Danvers, MA).