Sonifier 450 homogenizer

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).

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, USA). The samples were centrifuged at 12,000× g for 10 min at 4 °C. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Tissue supernatants (50 μg) were resolved by SDS-PAGE and subjected to immunoblotting using chemiluminescence detection (Thermo Fisher Scientific, Rockford, IL, USA) and quantified using Image J software after normalizing to β-actin or when appropriate, total protein kinase B (AKT) or total AMP kinase alpha subunit AMPKα. Nitrocellulose membranes were incubated with antibodies for 1–2 h at room temperature or overnight at 4 °C as indicated (Table S2).