Stain free imager

At the completion of the functional measurements, the individual skinned fiber segments were diluted with 10 μl of non-reducing Laemmli buffer (mM): urea, 4000; Tris, 250; 4% SDS (vol/vol); 20% glycerol (vol/vol); 0.02% bromophenol blue (wt/vol). In pilot experiments, we confirmed that the skinned fiber samples contain the remaining nondiffusible components (i.e., myofibrillar proteins), but not the diffusible (i.e., cytosolic) proteins (data not shown). To separate the myofibrillar proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using a 4–15% Criterion Stain-Free gel (Bio-Rad, Philadelphia, PA, United States). Images of the gels were acquired using Stain-Free imager (Bio-Rad). The ratio of myosin heavy chain (MyHC) to actin was evaluated densitometrically using ImageJ¹ (National Institute of Health, Bethesda, MD, United States). Then, the fiber type was determined by immunoblot using anti-fast type MyHC antibody (see the Section “Immunoblots”).

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)].

Myosin heavy chain (MyHC) and actin content was assessed using quantitative gel electrophoresis and immunoblotting, respectively (immunoblots for actin, see the section “Immunoblots”). To extract whole muscle proteins, muscle pieces were homogenized in ice-cold homogenizing buffer (40 μl/mg wet wt) consisting of: 10 mM Tris maleate; 35 mM NaF; 1 mM NaVO₄; 1% Triton X-100 (vol/vol); 1 tablet of protease inhibitor cocktail (Roche) per 50 ml. The protein content was determined using Bradford assay [32 (link)]. Whole muscle homogenates (2.0 μg total muscle mass) were diluted with SDS-sample buffer: 62.5 mM Tris/HCl; 2% SDS (wt/vol); 10% glycerol (vol/vol); 5% 2-mercaptoethanol (vol/vol); 0.02% bromophenol blue (wt/vol) and were loaded onto 4–15% Criterion TGX Stain Free gels (BioRad) together with known amounts of purified recombinant proteins (rabbit myosin and actin, Sigma), the latter allowing a calibration curve to be generated. Gels were imaged (BioRad Stain Free imager) and the density of MyHC was measured by using Image Lab Software (BioRad). When quantifying absolute amounts of MyHC and actin, the density of the relevant band was converted to an equivalent protein amount, according to the calibration curve derived from the pure protein samples run on the same gel. This amount was expressed relative to the muscle mass.