Cl caspase 3

Saos-2 cells were purchased in ATCC and cultured in conditioned medium containing 90% DMEM and 10% FBS. The antibodies of Bad (Abcam, ab32445), Bcl-2 (Abcam, ab692), cl-caspase 3 (Abcam, ab13585), CDC25C (Cell Signaling Technology, 4688), cyclin B1 (Cell Signaling Technology, 4138), CDK2 (Cell Signaling Technology, 2546), cyclin A (Cell Signaling Technology, 4656), GAPDH (Abcam, ab37168) were purchased.

OCT4 (#ab19857), Cl-caspase 3 (#ab32042), Cleaved-PARP (#ab32064), Bcl2 (#ab182858) and Bax (#ab32503) primary antibodies were obtained from Abcam (Cambridge, MA, USA). The transfected cells were washed twice with New phosphate-buffered saline containing 0.1% Tween (PBST) before extracting the total protein at 4 ℃ by using RIPA lysis buffer with protease inhibitors. We then centrifuged the lysates for 25 min at 15,000 rpm to remove the cell debris. The concentration of protein was measured using protein quantification kit (Thermo Fisher, Carlsbad, CA, USA). Fifty µg extracts of protein were added into each lane and then were fractionated by using 12% SDS-PAGE gel, and were then transferred onto the 0.22 µm PVDF membrane (Merck Millipore, Billerica, MA, USA). Subsequently, the PVDF membrane was blocked for 2 hours at radiotherapy (RT) using 5% non-fat milk as the blocking reagent, and was then incubated with the primary antibodies for 2 hours at RT. GAPDH was used as housekeeper control. Two hours later, the membrane was washed twice with Tris buffered saline with Tween 20 (TBST), and was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 hours. Finally, the signal of the target proteins was detected with enhanced chemiluminescence (ECL, Thermo Fisher).