Gapdh no 5174

Lysis of MC3T3-E1 cells took place in radio immunoprecipitation assay buffer with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd.), and centrifugation of lysates continued for 15 min at a temperature of 4°C at 9,600 × g so that precipitation can be removed. Subsequently, we assessed the amount of protein with the BCA assay kit (Thermo Fisher Scientific, Inc.). Equal amount of protein was added onto a 10 or 15% SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore, Bredford, MA, USA). The membranes were blocked with 5% skim milk and probed with primary antibodies. After washing 3 times with phosphate-buffered saline (PBS), incubation of membranes with horseradish peroxidase conjugated secondary antibody (1:1,000; cat no. A0208; Beyotime Institute of Biotechnology, Shanghai, China) was conducted. We conducted western blotting tests via an enhanced chemiluminescence (ECL) kit (EMD Millipore). GAPDH was used as the internal standard. The sources of primary antibodies were as follows: Bax (Sc-493) and Bcl-2 (Sc-492) both from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); p53 (no. 2524) and GAPDH (no. 5174) both from Cell Signaling Technology, Inc. (Danvers, MA, USA).