Ab133264

Protein lysates were prepared in the RIPA buffer (radioimmunoprecipitation assay lysis buffer) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Thermo Scientific, Waltham, MA). The total protein concentration of the soluble extract was determined using the BCA protein assay Kit (Thermo Scientific). Each protein sample (30μg) was resolved to SDS–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated overnight at 4 °C with primary antibodies. The antibodies used were ERG (1:2,000 Abcam, #ab133264); JUN (1:1,000 Santa Cruz, sc-1694X); rabbit anti-FLAG (1:1,000 Sigma, F7425); pan-cytokeratin (1:1,000 BioLegend, clone C-11 cat # 628602 and Lot # B181240); αtubulin (1:1,000 Sigma, #T6199) and GAPDH (1:10,000 Abcam, sb-75834). Following three washes with TBS-T, the blot was incubated with a horseradish peroxidase-conjugated secondary antibody and immune complexes were visualized by enhanced chemiluminescence detection (ECL plus kit, GE Healthcare, UK). The blot was reprobed with a monoclonal antibody against beta-actin (1:10,000 Sigma). Total protein was extracted and separated by gel electrophoresis. Protein was then transferred to nitrocellulose membranes and probed overnight using the appropriate primary antibodies.