Nupage 4 12 bis tris gel

Cells, human tumor samples, mouse normal mammary gland tissues, and mouse breast tumor samples were homogenized in 1× RIPA buffer (EMD Millipore) supplemented with Protease/ Phosphatase Inhibitor Cocktail (Pierce, Thermo Scientific), Panobinostat (Selleck chemicals), and Methylstat (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4 °C. Supernatants were analyzed for immunoblotting or immunoprecipitation with the indicated antibodies, and the immunoprecipitated proteins were subjected to either immunoblotting or protein identification by mass spectrometry. For the denaturing immunoprecipitation, tissues were lysed using TSD buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 5 mM DTT) boiled for 10 min. The cleared lysates were used for immunoprecipitation. The elutions were loaded on NuPAGE 4%–12% Bis-Tris Gel (GenScript) and then analyzed for immunoblotting with the indicated antibodies summarized in Supplementary Table 6.