Ab51068

Protein isolation was performed using working RIPA buffer (120mM NaCl, 50 mM pH 8.0 Tris, 0.5% NP-40, 1 mM EGTA) containing protease and phosphatase inhibitors (100 ug/mL PMSF, 1 mM NaOrVa, 50 ug/mL aprotinin, 50ug/mL leupeptin). Protein concentration was determined using a Bio-Rad protein concentration assay dye and absorbance read at 595 nm on a spectrophotometer (SpectraMax ID3, Molecular Devices). 20μg total protein in βmercaptoethanol-containing loading buffer was added per well onto 4–12% SDS-PAGE gels (NuPAGE) and transferred to PVDF (Bio-Rad) or nitrocellulose (Amersham) membrane. nitrocellulose membranes were stained for total protein using Ponceau S (Boston BioProducts). Membranes were blocked with 10% BSA or milk-fat in TBST for 1 hour at room temperature. Primary antibodies from Abcam for AR (ab74272), AR-V7 (ab198394), phosphorylated AR-Ser650 (ab47563), ACAT1 (ab168342), MAP3K11 (ab51068), PSDM12 (ab229930), and GAPDH (AM4300) were used at recommended concentrations in 2.5% milk-fat or 2.5% bovine serum albumin overnight at 4°C. Anti-rabbit and anti-mouse secondary antibodies (GE Healthcare) were used in 2.5% milk-fat for one hour at room temperature. Membranes were imaged via chemiluminescence reagents (Super Signal™ West Pico Plus, Thermo Fisher) on a digital imager (ChemiDoc™ Touch, Bio-Rad).