Irdye labeled anti rabbit or anti mouse secondary antibodies

Cells were lysed in nondenaturing RIPA buffer (50 mm Tris·HCl (pH 7.4), 150 mm NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 2 mm Na₃VO₄, 2 mm NaF) supplemented with protease inhibitors (Roche Applied Science). Lysates were incubated with protein A/G–Sepharose beads (Santa Cruz Biotechnology) and anti-FLAG or anti-HA tag antibodies overnight at 4 °C. Beads were collected, washed four times with RIPA buffer, and boiled in SDS loading buffer. Samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and incubated with primary antibodies overnight at 4 °C. After washing, blots were subsequently incubated with IRDye-labeled anti-rabbit or anti-mouse secondary antibodies (LI-COR Biosciences) and analyzed by Odyssey IR scanning (LI-COR Biosciences).

Cells were lysed in RIPA buffer (50 mM Tris·HCl [pH 7.4], 150 mM NaCl, 0.5% Triton X-100, 2 mM Na3VO4, 2 mM NaF) supplemented with protease inhibitors (Roche). Lysates were boiled in SDS loading buffer and subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and incubated with primary antibodies in 5% BSA overnight at 4°C. After washing, blots were subsequently incubated with IRDye-labeled anti-rabbit or anti-mouse secondary antibodies (LI-COR Biosciences) and analyzed by ChemiDoc imaging system (Bio-Rad). The following primary antibodies were used in this study: anti-4E-BP1 (Cell Signaling, 53H11), anti-PTEN (Cell Signaling, 138G6), anti-phospho-Akt^S473 (Cell Signaling, D9E), anti-Akt (Cell Signaling, C67E7), anti-alpha-Tubulin (DSHB, 12G10), and anti-phospho-4E-BP1^S83 (Millipore, ABE2889).