α 4e bp1

Samples for immunoblots of mammalian proteins were prepared by 3 × wash with cold 1 × PBS and kept at −80 °C until 2 × Laemmli buffer was added to make whole cell lysates. Total cell extracts for immunoblots of S. pombe proteins were made by TCA protein extraction^{53 (link)}. Antibodies for immunoblots were α-γtubulin (1:10000, T6557, Sigma-Aldrich), α-CHK1-P345 (1:1000, 2348, Cell Signaling Technology), α-CHK1 (1:200, DCS310.1, Santa Cruz), α−4EBP1 (1:2000, Cell Signaling Technology) α-phosphoAkt substrates (1:1000, 23C8D2, Cell Signaling Technologies), α-PSTAIRE, recognizing a motif in cdc2 (1:2000, Santa Cruz Biotechnology sc-53) and anti-peroxidase PAP1, against the TAP-tap (1:1000, Sigma P1291). Appropriate ECL and ECF kits were used for detection.

Gastrocnemius muscle from 4-month-old mice was homogenized in cold SDS lysis buffer (50 mM Tris, pH 7.5, 70 mM urea, 250 mM sucrose, and 2% SDS) with Protease Inhibitor Cocktail (Roche, 4693159001) and Phosphatase Inhibitor Cocktail II (Sigma, P5726) and III (Sigma, P0044). 30 μg of total protein was separated in 10% polyacrylamide gel (Bio-Rad, #1610158) and transferred to nitrocellulose membranes. Ponceau red (MP Biomedicals, 02190644) was used to check the transfer efficiency and also applied to the total protein loading. After washing out the ponceau red staining, the membrane was blocked with 5% milk for an hour. The following antibodies from Cell Signaling were used for Western blotting: α- p70 S6 kinase 1 (9202), α- p70 S6 kinase 2 (14130), α-S6 Ribosomal Protein (2217), α-phospho-S6 ribosomal protein Ser240 + 244 (2215), α− 4EBP1 (9452), α phospho-4EBP1 Thr37 + 46 (2855), α- Akt (4691), α phospho-AKT Ser473(4060) and α-actinin (3134). α phospho-p70 S6 kinase 2 Ser423 (SAB4301595) was purchased from sigma and α TSC1 (A300-316A) was purchased from Axil Scientific.