Mab150

Worms were synchronized by hypochlorite extraction of eggs and exposed to control or experimental conditions beginning on day 2 of adulthood. Protein was extracted 24 h later by 3 rounds of freeze-thaw cracking and dounce homogenization in a homogenization buffer [15 mM Hepes (pH 7.6), 10 mM KCl, 1.5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM EGTA 44 mM, 1 mM DTT, 2 mM NaVO₄, 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, 1x Phosphatase Inhibitor Cocktail 2 (Sigma)]. Samples were run on a 4–12% Bis-Tris gel in MOPS buffer and transferred to a PVDF membrane. Blots were blocked for 15 min in 5% nonfat dehydrated milk and probed for 1 h with either a mouse α-HIF-1 antibody in whole serum provided by Dana Miller (Budde and Roth, 2010 (link)) or mouse α-actin antibody MAB150 (Millipore) diluted 1:10000 in 1% milk. Santa Cruz Biotechnology secondary HRP-conjugated goat α-mouse IGG antibody (sc-2064) was diluted 1:5000 in 1% milk and incubated for 1 h. SuperSignal chemiluminescent reagents (Thermo Scientific) were used as HRP substrate according to the manufacturer's recommended procedure. Abcam ab69312 and Santa Cruz Biotechnology sc-9996 were used at a 1:2500 dilution for anti-GFP immunoblotting.

Anti-actin antibody, clone C4 (Millipore Sigma MAB150), α-actinin (sarcomeric) antibody (Millipore Sigma A7732), anti-myosin (Skeletal, Slow) (Millipore Sigma M8421), and GAPDH antibody (G-9) (sc-365062; Santa Cruz, Dallas, TX).