Phosphorylated akt473

Proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Nonspecific proteins were blocked with 5% nonfat dried milk in Tris-buffered saline Tween (TBST) buffer for 1 h at room temperature with agitation and then membranes were incubated with various primary antibodies. These were monoclonal mouse anti-Cx43 (1:1000; Invitrogen, Carlsbad, CA), monoclonal rabbit anti-Akt (1:1000; Cell Signaling Technology, Beverly, MA), phosphorylated Akt⁴⁷³ (1:1000; Cell Signaling Technology), or rabbit anti-GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4–8 °C. After washing 3 times for 10 min per times in TBST, each membrane was incubated with the homologous secondary antibody for 1 h at room temperature. The images were analyzed using Quantity One software (Bio-Rad, Hercules, CA) to obtain the integrated intensities. Each experiment was repeated five times.

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.