Western Blot Analyses

Loading for immunoblotting was determined to be 15, 10, and 5 μg/sample for citrate synthase, oxidative phosphorylation (OXPHOS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) proteins, respectively. A biotin-conjugated molecular weight marker (Cell Signaling, Beverly, MA, United States) was used to confirm the target proteins by size. Samples were loaded according to muscle type and included a representation of all treatment groups for each gel. Protein was denatured by heating samples in SDS–PAGE sample buffer (0.2% SDS, 20% glycerol, 25% 4X buffer, 5% β-mercaptoethanol, and 0.025% bromophenol blue) at 42–44°C for 3 min and electrophoresed in an SDS-12% polyacrylamide gel at 85 V for 20–25 min and then 135 V for 80 min. The proteins were transferred to PVDF membranes for 3 h at 60 V (∼425 mA total). Next, the membranes were placed in a solution of Ponceau S (Sigma) to verify that protein loading was similar between samples and that the transfer was uniform and artifact-free (data not shown) before immersed in a blocking solution containing 5% nonfat dry milk (NFM) dissolved in phosphate buffered saline with 0.05% Tween-20 (T-PBS) for a minimum of 0.5 h. All antibodies were diluted in blocking solution and the membranes were incubated in either rabbit anti-GAPDH (1:30,000; Proteintech, Rosemont, IL, United States), rabbit anti-citrate synthase (1:2,000; ab96600, Abcam, Cambridge, MA, United States), or mouse anti-OXPHOS (1:40,000; Abcam) antibodies for 1 hr at room temperature. The primary OXPHOS antibody (ab110413) is a five-in-one mouse monoclonal antibody cocktail containing: a subunit of mitochondrial NADH (NDUFB8, ab110242); the iron-sulfur subunit of mitochondrial succinate dehydrogenase (SDHB, ab14714); mitochondrial cytochrome b-c1 complex subunit 2 (UQCRC2, ab14745); mitochondrial-encoded cytochrome c oxidase I (MTCO1, ab14705); and the alpha subunit of mitochondrial ATP synthase (ATP5A, ab14748). The membranes were washed for 1 h in several changes of T-PBS and then incubated for 1 h at room temperature in horseradish peroxidase-linked secondary antibodies including, anti-mouse IgG (1:10,000) for OXPHOS targets, anti-rabbit IgG for citrate synthase (1:15,000) and 1:20,000 for GAPDH targets, and anti-biotin (1:1,000) the molecular weight marker. Finally, the membranes were washed for 1 h in T-PBS and then developed using an ECL detection kit (Amersham, Piscataway, NJ, United States) per the manufacturer’s instructions. Exposure to Kodak X-Omat film was either ∼5, 10–12, or 35–40 s for GAPDH, citrate synthase, or OXPHOS targets, respectively. For each protein, densitometry and quantification were performed using ImageJ. The adjusted relative density was ascertained using GAPDH as the loading control for each OXPHOS protein, averaged, and compared between groups.