Criterion tgx gradient

The immunoblotting process was performed as described in ref.^{34 (link)} with minor modifications. The amount of protein loaded in each well is specified below for the antibody validation experiments, while 20 and 2.5 µg of protein were loaded for the whole muscle homogenate and fiber type pool specific analyzes, respectively. Both 18- and 26-well Criterion TGX gradient (4%-20% acrylamide, Bio-Rad Laboratories, Richmond, CA, USA) were used for electrophoresis. Finally, all antibodies were probed on membranes on first use, and no stripping or re-probing were performed. Details about the antibodies used for immunoblotting are presented below.

For Western blot analyses, protein samples were prepared and analyzed as described before [4 (link)]. Briefly, proteins were isolated using RIPA buffer, and 25 µg per sample was separated by SDS-PAGE using 4–15% Criterion TGX gradient gels (BioRad Laboratories, Munich, Germany). Proteins were transferred to a nitrocellulose membrane by wet blotting and protein imaging was performed using UV transillumination on a Chemidoc XRS (Bio-Rad Laboratories). For the detection of target protein-specific primary antibodies [mTOR (7C10) Rabbit mAb, # 2983, 1:1500; Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb, # 5536, 1:2000; AKT (pan) (C67E7) Rabbit mAb, # 4691, 1:10,000; Phospho-AKT (Ser473) (D9E) XP Rabbit mAb, # 4060, 1:5000, each Cell Signaling Technology, Danvers, MA, USA] and a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, HRP-linked Antibody, Cell Signaling, #7074, 1:5000) were used. The relative protein abundance was visualized by enhanced chemiluminescence detection using an image-capture system (Chemidoc XRS, BioRad Laboratories). For normalization, the total protein loading was detected by the UV transillumination of membranes.