S6 ribosomal protein

Western blot was performed as described previously [10 (link)]. Briefly, proteins from tissues or cells were extracted and quantified. Aliquots of 10 μg total protein of each sample were separated by 8%-15% SDS-PAGE and transferred to nitrocellulose membrane for all the proteins except for LC3, where PVDF membranes were used for LC3 signal. The membrane was blocked in 5% skimmed milk-TBST solution for 1 hour at room temperature. The membrane was probed with primary antibodies at 4°C overnight. Antibodies to Akt, phosphor-Akt at Ser473 (p-Akt), mTOR, phosphor-mTOR at Ser2448 (p-mTOR), LC3A/B, p62, ribosomal protein S6, ribosomal protein phosphor-S6 at Ser235/236 (p-S6), and Na/K-ATPase were purchased from Cell Signaling Technology. The antibodies to glucose transporter 4 (GLUT4) (H-61) and EIF-5 were bought from Santa Cruz Biotechnology (CA, USA). EIF-5 was measured as an internal control of total protein. Na/K-ATPase was used as a loading control of plasma membrane protein. The membrane was washed for 5 min with TBS-T buffer for 5 times and then incubated with a horseradish peroxide-conjugated secondary antibody at room temperature for 1 h. After washing, the membrane was developed with ECL Reagent (Millipore, USA) and exposed to Kodak XBT-1 film. Protein expression level was quantified with Image J (NIH) software.

Myoblasts were grown on 6-well plates as described, washed in PBS, lysed, and scraped in 150 mL RIPA (Pierce/Thermo Scientific Waltham, MA USA, 89900) supplemented with PhosSTOP (Roche Penzberg, Germany, 04 906 837 001) and 1% protease inhibitor cocktail (Sigma P8340). Cells were lysed at 4°C for 20 minutes, centrifuged at 12,000g for 15 min, and the pellet was discarded. Protein concentrations were measured by BIO-RAD DC assay, and 5–10 mg of protein was run. Primary antibodies and dilutions: DUX4 E55 1:1000 (AbCam, Cambridge, UK, ab124699), Vinculin 1:10,000 (Sigma V9131), phosphorylated ribosomal protein S6 1:1000 (Cell Signaling Danvers, MA USA, 4858S), ribosomal protein S6 1:1000 (Cell Signaling 2317S), phosphorylated AKT 308 1:1000 (Cell Signaling 13038P), phosphorylated AKT 473 1:1000 (Cell Signaling 4060S), Pan-AKT 1:1000 (Cell Signaling 4691P), ULK1 1:1000 (Cell Signaling 8054T/S), and LC3A/B 1:1000 (Cell Signaling 12741T). Secondary antibodies and dilutions: anti-rabbit and anti-mouse IgG-HRP 1:2000 (Cell Signaling 7074P2 and 7076S). ECL reagents: Clarity Western ECL Substrate (BIO-RAD, Hercules, CA 102031761) and Clarity MAX Western ECL Substrate (BIO-RAD 76SF121P). Blots were visualized on a BIO-RAD ChemiDoc. Uncropped versions of all western blots are presented in the supplementary materials.

Proteins of the membrane-enriched fraction were loaded on acrylamide/bisacrylamide (29:1) gels and electrotransferred to nitrocellulose membranes, except for LC3 analysis, for which proteins were electrotransferred to polyvinylidene fluoride membranes. Antibodies against the following proteins were used for western blot analysis: Beclin-1 (Cell Signaling Technology, 3738), FYCO1 (Novus Biologicals, NBP1-47266), LAMP-2 (Abcam, ab13524), LC3 (Cell Signaling Technology, 2775), p62 (Sigma Aldrich, P0067), ribosomal protein S6 (Cell Signaling Technology, 2217), and ubiquitinated proteins (Enzo Life Sciences, BML-PW8810). Signals were revealed with the Clarity Western ECL substrate (Bio-Rad) and acquired with the ChemiDoc Touch Imaging System (Bio-Rad). Signals were quantified with the Image Lab Software (Bio-Rad).