Tissue Lysis and Protein Quantification

Skeletal muscle and liver lysates were prepared from powdered tissue by homogenizing in 25 mM HEPES, pH 7.4, 1% Igepal CA630, 137 mM NaCl, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 10 mM Na₄P₂O₇, 100 mM NaF, and 2 mM NaVO₄ using a Sonifier 450 homogenizer (VWR, Radnor, PA). The samples were centrifuged at 14,000×g for 10 min at 4 °C. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions. The tissue supernatants (50 μg) were resolved by SDS-PAGE and subjected to immunoblotting using chemiluminescence detection (Thermo Fisher Scientific, Rockford, IL) and quantified as described [47 (link)]. Nitrocellulose membranes were incubated with antibodies for 1–2 h at room temperature or overnight at 4 °C as indicated. An additional file provides detailed information about each antibody used (see Additional file 1).