Criterion stain free gel

Immunoblotting of single fibers and wash solutions was performed by separating protein on 4–15% Criterion Stain‐Free gels (Bio‐Rad, Hercules, CA) as previously described (Murphy et al. 2012). Following transfer, nitrocellulose membranes were blocked and probed with GLUT4 antibody while being constantly rocked overnight at 4C and 2 h at room temperature. Following washes and exposure to the secondary antibody (goat anti‐rabbit IgG‐HRP, 1:20000, 31460, Pierce), chemiluminescent images were captured and quantified using densitometry (Chemidoc MP and ImageLab software, Bio‐Rad, Hercules, CA). The proportion of GLUT4 protein in a given pool was expressed as a percentage of the total GLUT4 in all four of the single fiber fractions, that is, %GLUT4 in Trit = GLUT4 density in Trit/sum densities GLUT4 (Diff + Amy + Trit + FSk).

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)].