Rabbit anti phospho p70 s6 kinase

Aliquots of the cells harvested after transfection were mixed with sample buffer, consisting of 0.01% (w/v) bromophenol blue, 2% (w/v) SDS, 5% (w/v) β-mercaptoethanol, 25% (v/v) glycerol and 62.5 mM Tris-HCl (pH 6.8) and boiled for 10 min. Then, 10 µg of protein from the cell samples were loaded onto SDS polyacrylamide gels, electrophoresed and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk for 60 min at room temperature and then incubated overnight at 4°C with the primary antibodies. The primary antibodies were as follows: rabbit anti-Phospho-mTOR (1:1000 dilution; Ser2448, Cell Signaling), rabbit anti-mTOR (1:1000 dilution; Cell Signaling), rabbit anti-Phospho-p70 S6 Kinase (1:1000 dilution; Ser371, Cell Signaling), rabbit anti-Phospho-4E-BP1 (1:1000 dilution; Cell Signaling), rabbit anti-BCL2 (1:2000 dilution; Proteintech) and mouse monoclonal IgG1 antibody specific for β-actin (1:5000 dilution; Santa Cruz Biotechnology). After incubation with the primary antibodies, the membranes were washed four times with Tris-buffered saline containing 0.1% Tween-20 and incubated with the secondary antibodies (1:2000 dilution; Cell Signaling) for 90 min at room temperature. The ECL chemiluminescence kit (Millipore) was used to detect the protein bands.