HIF-1α protein expression and two of its target proteins, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1), were semi-quantified using Western blot technique. Whole GAS muscles were homogenized in urea lysis buffer [6 M urea, 1% sodium dodecyl sulfate (SDS)], supplemented with protease and phosphatase inhibitors (Complete Protease Inhibitor Cocktail and PhosphoSTOP, Sigma-Aldrich), using the Precellys® Evolution tissue homogenizer (Bertin Technologies, Montignt-le-Bretonneux, France). The lysates were centrifuged at 25,000 g for 15 min at 4°C to remove cell debris, and total protein content of the collected supernatants was quantified by the bicinchoninic acid assay (Thermo Fisher Scientific). Muscle protein extracts were solubilized in an electrophoresis loading buffer (62.5 mM Tris⋅HCl, pH 6.8; 2.3% SDS; 10% glycerol; 5% mercaptoethanol; and bromophenol blue), and equal amounts of protein (between 10 and 55 μg) were loaded onto each lane of the gel, and electrophoresis was run on 10% SDS–polyacrylamide gel electrophoresis (PAGE) gels. Ten study samples and one control sample in triplicate (to verify the possible variability between samples across each membrane) were loaded onto each gel. The following internal quality control criteria were used to consider a valid blot: the variation between the control samples had to be less than 20%, and the same control sample was loaded in all the gels to compensate for the variability between gels and to be able to compare them, as described by Martin-Rincon et al. (2019).

After electrophoresis, proteins were transferred to an Immun-Blot polyvinylidene fluoride (PVDF) membrane for protein blotting (Bio-Rad Laboratories). To verify that equal amounts of muscle protein were charged in each well and the efficiency of transference, the membranes were stained with Ponceau S stain (Sigma-Aldrich). For immunoblotting, membranes were blocked with 4% bovine serum albumin (BSA) in TBS-T (Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature. To detect our target proteins, membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (MA1-516; Thermo Fisher Scientific), VEGF (MA1-16629; Thermo Fisher Scientific), and GLUT1 (#12939; Cell Signaling Technology) diluted 1:1,000 in 4% BSA-TBS-T. Following this, the membranes were incubated with the corresponding secondary horseradish peroxidase (HRP)-conjugated antibody (#31460 and #31430; Thermo Fisher Scientific) diluted 1:5,000 in 5% Blotto in TBS-T for 1 h at room temperature. The specific bands were visualized with the Clarity TM Western ECL Substrate Kit (Bio-Rad Laboratories), and the chemiluminescence signal was measured using the Odyssey Fc Imaging System (LI-COR Inc. Biotechnology, Lincoln, Nebraska, United States) and quantified with the Image Studio Software (v. 5.2.5, LI-COR Inc. Biotechnology). Muscle signaling data were reported (in arbitrary units) as the sample band intensity relative to Ponceau S stain, and the final data were normalized to the mean value (band densities) of the three control samples loaded on all the gels.

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