Amersham ecl anti mouse na 931

Cell protein lysates were harvested from osteoclasts grown in 12 well dishes (Corning) in modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were cleared by centrifugation at 12,000Xg at 4°C. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore), subjected to western blotting by standard protocols, and visualized using ECL Prime (G.E. Health Systems). Blots were blocked and incubated at 4°C with primary antibodies diluted in TBS/0.1% Tween-20 plus 3% bovine serum albumin. Primary antibodies used were HDAC7-Abcam (Ab12174) 1:2000 dilution; ^α-TUBULIN- Santa Cruz (SC-5546) 1:1000 dilution; MYC- Santa Cruz (SC-40) 1:1000 dilution; MITF- Abcam (Ab12039) 1:1000 dilution; ACTIN- Santa Cruz (SC-1616) 1:2,000 dilution; FLAG—Sigma-Aldrich (F1804) 1:5,000 dilution. Horseradish-peroxidase conjugated secondary antibodies were diluted in TBS/0.1% Tween-20 plus 5% nonfat dry milk. Secondary antibodies used were from G.E. Health Systems: Amersham ECL anti-mouse (NA-931) and anti-rabbit (NA-934) at 1:8000, or Santa Cruz: anti-goat (SC-2020) at 1:12,000 dilution.