Anti abcg2

Protein extracts of mouse ventricles or fibroblasts were prepared in RIPA buffer containing protease-inhibitor and phosphatase-inhibitor tablets (Roche, Vienna, Austria). After mechanical disruption of tissue samples, equivalent amounts of protein were separated on a SDS polyacrylamide gel, followed by electro-transfer to nitrocellulose membrane. Nonspecific antibody binding was blocked by incubation in 5% (m/v) non-fat dry milk powder in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mMNaCl, 0.1% (v/v) Tween 20) at room temperature for 1 h. Then, samples were incubated overnight at 4°C with antibodies: anti-ABCG2 (#ab3380, Abcam) and anti-α-tubulin (#ab4074, Abcam) for ventricular tissues, and anti-ABCG2 (#AV43649, Sigma-Aldrich, Saint Louis, USA) and anti-GAPDH (#sc-25578, Santa Cruz Biotechnology, Heidelberg, Germany) for fibroblasts. After 1 h incubation in room temperature with peroxidase-labeled secondary antibody (Pierce Biotechnology, Rockford, USA), specific immunoreactive signals were detected using Enhanced ChemiLuminescence kit (Amersham Biosciences, Buckinghamshire, UK) or West Pico Substrate (Thermo Scientific, Rockford, USA).

The lysates were prepared by RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) from U2OS cells (1 × 10⁶) or OSLCs (1 × 10⁶). The protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis with sodium dodecyl sulfate-polyacrylamide gel (10%, SDS-PAGE) was used to separate the lysate (40 μg protein), which was then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were then blocked with TBST containing 5% BSA for 2 h at room temperature and then incubated with primary antibodies anti-β-actin (1 : 5000; catalog no. A5441; Sigma-Aldrich), anti-CD133, anti-CD44, anti-ABCG2, anti-DNMT1, anti-Bmi1, anti-Sox2, and anti-OCT4 (1 : 2000; catalog nos. 5860S, 3570S, 4477S, 3598S, 5856, 2748, and 2840 CST) overnight at 4°C. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (Beyotime Institute) for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit (Amersham Biosciences) by using an enhanced chemiluminescence detection system (Ranon GIS-2008, Tanon Science & Technology Co., Ltd., Shanghai, China).