Amersham ecl chemiluminescence detection system

Cells were lysed using RIPA buffer (Sigma) containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma). Cell lysates were centrifuged at 12,000g for 10 minutes at 4 C. Total protein concentrations were measured using Bradford assay (Thermo Fisher Scientific, USA, http:// www.thermofisher.com), and equal amount of protein was loaded on a 10% polyacrylamide gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Blotted Polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4 C with antibodies against P-Akt (Ser473, Cell Signaling, Beverly, MA, http://www.cellsignal.com), P-RhoA (Ser188, Abcam, Cambridge, U.K., http://www.abcam.com), p-mTOR (Ser2448, Cell Signaling, USA, http://www.cellsignal.com), and Actin (Sigma). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) for 45 minutes at room temperature, and protein bands were visualized using Amersham ECL chemiluminescence detection system (GE Healthcare Bio-Sciences, USA, http://www.gelifesciences.com). Western blot band intensities were measured by image J and presented as relative to the control. All antibodies were used at a 1:1,000 dilution in 5% blotting grade milk solution prepared in PBST (PBS supplemented with 0.1% Tween 20).