Ab13847

Total protein was extracted from cells using M-PER buffer solution (Pierce Biotechnology Inc., Rockford, IL, USA) containing protease inhibitor (Roche, Basel, Switzerland) and Nonidet-P40. The protein concentration was then determined by the modified Coomassie protein assay (Bradford method). Then 50–80 μg of protein was separated using 10% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Next, the membrane was treated with 5–10% skim milk powder or 5% bovine serum albumin (BSA) followed by overnight incubation with primary antibodies against Bcl-2-associated X protein (Bax) (1:1,000, sc-7480), B-cell lymphoma 2 (Bcl-2) (1:1,000, sc-7382), YY1 (1:1,000, sc-7341), telomerase reverse transcriptase (TERT) (1:1,000, sc-393013), p53 (1:1,000, sc-126), p300 (1:2,000, sc-48343), GAPDH (1:1,000, sc-47724), caspase-3 (1:500, ab13847), and cleaved caspase-3 (1:500, ab13847) [all from Abcam Inc. (Cambridge, UK) except GAPDH which was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA)]. The following day, the membrane was re-probed with horseradish peroxidase-labeled secondary antibody for 1 h after Tris-buffered saline Tween-20 (TBST) washing. Afterwards, the membrane was visualized using enhanced chemiluminescence (ECL) reagents (Pierce), and the protein bands were quantified by Image Lab 3.0 software (Bio-Rad, Inc., Hercules, CA, USA).

Culture medium was removed, and cells were washed thrice with PBS. After 0.1% Triton X-100 and 3% H₂O₂ treatment, cells were incubated in rabbit polyclonal anti-Caspase-3 antibody (ab13847, Abcam, UK) at 4 °C overnight and anti-rabbit universal immunoassay kit (GeneTech, China) at 37 °C for 30 min. The cells were counted in five random fields per well. For BMSCs grafts evaluation, rats were sacrificed at 7 days after transplantation and the femoral trochlea were harvested. Frozen sections were stained by anti-Caspase-3 antibody (ab13847, Abcam, UK) at 4 °C overnight, and anti-rabbit immunoassay kit (GeneTech, China) at 37 °C for 1 h. Each frozen section was counted in five random fields.

Western blot analysis was conducted as previously described [27 (link)]. The primary antibodies against proliferating cell nuclear antigen (PCNA; ab29; 1:1000), total-caspase 3 (ab13847; 1:500), cleaved-caspase-3 (C-caspase 3; ab13847; 1:1000), N-cadherin (ab202030; 1:1000), Vimentin (ab193555; 1:1000), E-cadherin (ab1416; 1:1000), FHL2 (ab12327; 1:1000) and GAPDH (ab8245; 1:1000), and the secondary antibodies, horseradish peroxidase (HRP) conjugated goat anti-rabbit (ab205718; 1:5000) and goat anti-mouse (ab205719; 1:5000), were purchased from Abcam (Cambridge, MA, USA).