Ezh2 ab186006

Protein extraction and cell lysates were prepared using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Transgen Biotech, China). GAPDH (ZSJQB Co., Ltd., Beijing) was used as a control. BRMS1 (ab134968) and EZH2 (ab186006) were purchased from Abcam.

MCF-7 or MDA-MB-231 cells were harvested and protein was extracted using radioimmunoprecipitation buffer (50 mM tris-HCl pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl and 0.1% SDS). The Bradford assay reagent (Thermo Fisher Scientific, Inc.) was then used to determine the protein concentration in the lysates. Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE gel, and then transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk in PBS containing 0.5% Tween-20 at room temperature for 1 h and incubated with the primary antibodies overnight at 4°C, and then washed three times with washing buffer Tris-buffered saline Tween-20 (Sigma-Aldrich, Merck KGaA). Horseradish peroxidase-conjugated anti-rabbit (sc-2357; 1:3,000) or anti-mouse (sc-2789, 1:3,000; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies were used as the secondary antibodies at room temperature for 1 h. The signal was visualized using enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific, Inc.). The primary antibodies used were as follows: SOX5 (ab94396; 1:1,000), EZH2 (ab186006; 1:1,000; both Abcam). β-actin (sc-47778; 1:1,000; Santa Cruz Biotechnology, Inc.) was used as the control.