Protease phosphatase inhibitors cocktail

Total skeletal muscle or CD11b⁺ infiltrating cells were lysed in 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 10 mM EDTA and protease/phosphatase inhibitors cocktail (Sigma-Aldrich). Lysates were centrifuged at maximum speed for 15 min at 4 °C. For western blot analysis, equal amounts of protein (20 μg) were resolved by SDS polyacrylamide gel electrophoresis and transferred onto Immobilon-P (Merck Millipore). After Ponceau S (Sigma-Aldrich) staining, membranes were saturated in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl (Tris-buffered saline) containing 2% non-fat milk and 0.1% Tween 20. Antigens were detected using the following antibodies: rabbit anti-mouse HIF1-α (Cayman Chemicals, Ann Arbor, MI, USA), mouse monoclonal anti-TGF-β (clone 1D11, Bio x cell, West Lebanon, NH, USA), mouse monoclonal anti-β-actin (Sigma-Aldrich), mouse monoclonal anti-GAPDH (Sigma-Aldrich). All antibodies were diluted in TBST 2% not-fat milk. Immunoreactive bands were revealed using an ECL detection kit (GE Healthcare Europe GmbH, Little Chalfont, UK).