Anti hif 1α

Skeletal muscles were homogenized in a lysis buffer (50 mM Tris-HCl, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM DTT, 1 mM Na₃VO₄, 10 mM NaF, 10 mM NaPPi, and 10 mM NaMo) and centrifuged at 20,000 × g for 15 min at 4°C. C2C12 myotubes were sonicated in a lysis buffer and centrifuged at 20,000 × g for 15 min at 4°C. The supernatants from homogenates and lysates were subjected to SDS-PAGE and analyzed by Western blot analysis using mouse monoclonal antibodies [anti-MyHC I (clone BA-D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), anti-MyHC IIB (clone BF-F3; Developmental Studies Hybridoma Bank, University of Iowa), and anti-β-actin (clone 2D4H5, Proteintech Group, Inc., Chicago, IL) antibodies], and rabbit polyclonal antibodies [anti-HIF-1α (Bethyl Laboratories; Montogomery, TX) and anti-CD36 (Novus Biologicals; Littleton, CO) antibodies]. The immunoreactive proteins were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (BioRad, Hercules, CA) and anti-mouse IgG (BioRad) and reacted with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA), followed by detection with an LAS4000 imaging system (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK).