Quantification of Muscle Protein Levels

The protein levels were quantified using cryosectioned slices of frozen soleus and EDL muscles using Western blotting procedures, as described in detail previously [39 (link),40 (link),41 (link)]. Proteins assessed included CSQ1, SERCA1, GLUT4, GS, glycogen branching enzyme (GBE), glycogen phosphorylase (GP), GDE, and COXIV as described by [40 (link)], with DHPR and RyR1 as described by [41 (link)]. In brief, proteins, including 4–5 amounts of a mixed muscle homogenate used as a calibration curve, were separated on 4–15% Criterion Stain Free gels (Bio-Rad, Hercules, CA). After transferring to nitrocellulose and a series of washes, including antibody probes, protein bands were visualized using West Femto chemiluminescent substrate (Thermo Scientific, Carlsbad, CA, USA), images collected, and densitometry performed using Chemidoc MP system and Image Lab version 5.2 (Bio-Rad, Hercules, CA, USA). Total protein on the gels was imaged prior to transfer (Stain Free imager, Bio-Rad) and Western blot signals of given proteins normalized to total protein with both total protein and protein of interest expressed relative to their respective calibration curves [39 (link)].