Tris glycine gels with tris glycine sds buffer

Western blot analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in RIPA buffer (Sigma; 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with Complete Protease Inhibitor Cocktail (Roche) and 1 mM PMSF. Proteins were separated by SDS-PAGE (Tris-glycine gels with Tris/glycine/SDS buffer, Bio-Rad) and transferred onto nitrocellulose membranes using the Trans Turbo Blot system (Bio-Rad). Membranes were probed with specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The following primary antibodies were used: FOXO1 (Cell Signaling Technology, #2880, 1:1000), pThr24FOXO1/pThr32FOXO3a (Cell Signaling Technology, #9464, 1:1000), c-MYC (Cell Signaling Technology, #9402, 1:1000), Tubulin (Cell Signaling Technology, #2148, 1:1000), Secondary antibodies are peroxidase-conjugated Goat IgGs (1:5000) purchased from Jackson Immuno Research Labs. The target proteins were visualized by chemiluminescence using an ECL detection kit (Clarity Western ECL Substrate, Bio-Rad) and a ChemiDoc MP Imaging System (Bio-Rad).