Immunoblotting of Phosphorylated Proteins

Tissue samples were collected and flash frozen. After 24 h, protein was extracted and immunoblotting performed as previously described.^{43 (link)} The membranes were blocked with 5% non-fat dry milk for 1 h and then probed with primary antibodies against phospho RPS6 (Ser235/236, 4858, Cell Signaling Technology), phospho AKT (Ser473, 4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, 2855, Cell Signaling Technology), total RPS6 (2217, Cell Signaling Technology), total AKT (4691, Cell Signaling Technology) or total 4EBP1(9644, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH antibody (8884, Cell Signaling Technology) was used as a loading control at a ratio of 1:5000.