2.8. Protein Analyses
PGC-1α, human skeletal muscle
LYSED, SECONDARY
The content of specific proteins involved in energy production in human skeletal muscle was measured by western blot analysis. Muscle was lysed in buffer that contained 20 μL of Radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA, USA) for every 1 mg of muscle sample and 3 inhibitors (Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 2 and Phosphatase Inhibitor Cocktail 3, Sigma-Aldrich, St. Louis, MO, USA) at a proportion of 2% in the total mixture of the protein extract. Muscle samples were homogenized using a Qiagen TissueLyser II (Qiagen, Hilden, Germany). Samples were spun at 15,000× All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), including 5′ AMP-activated protein kinase alpha (AMPK-α) (2532), phospho-AMPKThr172 (2535), acetyl-CoA carboxylase (ACC-α; 3662), phospho-ACCSer79 (3661), and PGC-1α (2178). A secondary antibody (Anti-Rabbit IgG (Goat), HRP-Labeled) was purchased from PerkinElmer (Melbourne, Australia). The volume density of each target band was normalized to total protein loaded into each lane using stain-free technology (Bio-Rad Laboratories, Hercules, CA, USA). After protein loading normalization, each phosphoprotein was then normalized to its respective total protein. Analysis of the western blots was performed using the Image Lab software 6.0.1 (Bio-Rad Laboratories, Hercules, CA, USA). The content of each protein was quantified using the Image J software 1.53 (National Institute of Health, Bethesda, MD, USA).