Ripa buffer

The cells were lysed in RIPA buffer (Macgene, Beijing, China, #MP015) supplemented with a protease inhibitor (MedChemExpress, Monmouth Junction, NJ, USA, HY-K0012). After determining the protein concentration using a BCA Protein Assay Kit (Thermo Fisher Scientific, Bremen, Germany, #23227), equivalent protein quantities were subjected to SDS–PAGE and then transferred to PVDF membranes (Millipore, Darmstadt, Germany, #GVWP02500). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with the indicated primary antibodies, followed by the appropriate HRP-conjugated anti-mouse/rabbit secondary antibodies (Zsgb, Beijing, China, #ZB-2301). Immunoreactive bands were measured with an enhanced chemiluminescence western blotting system (Millipore, Darmstadt, Germany, #WBKLS0500). For the detection of ATX, a secreted protein, cells were cultured in serum-free medium. The medium was concentrated (20-fold) using an Amicon Ultra 30000 (Merck KGaA, Darmstadt, Germany), and then subjected to SDS-PAGE and western blotting.

Cell pellets were lysed in RIPA buffer (Macgene Technology Ltd.) with 10 μl/ml protease inhibitor cocktail (P8340, Sigma) and 10 μl/ml phosphatase inhibitor cocktail (p0044, Sigma). The proteins in cell lysates were separated by electrophoresis on a 15% SDS–polyacrylamide gel, transferred to nitrocellulose membranes, immunostained, and visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China). Antibodies against Survivin, Bcl-2, Bcl-xL, P-Histone 3, phospho-Survivin, and phospho-Bcl-2 were purchased from Cell Signaling (Danvers, MA), and the antibody specific for phospho-Ser-62-Bcl-xL was purchased from Abcam (Cambridge, UK). The β-Actin antibody was purchased from Zsbio (Beijing, China).