Bcl2 associated x (bax)

The MSCs were seeded into a 60 mm petri dish, at a density of 10×10⁵ cells/dish. The total protein was extracted using radioimmunoprecipitation buffer (EMD Millipore), supplemented with PMSF. The cells were sonicated briefly and centrifuged at 10,000 x g at 4°C. The protein concentration was measured using the BCA Protein Assay kit, according to the manufacturer's instructions. Equal samples of protein (20 µg) were separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with blocking solution (Beyotime Institute of Biotechnology, Jiangsu, China) at room temperature for 2 h, and then incubated with the following primary antibodies: Bcl-2, Bax, caspase-3 and GAPDH (cat. no. KC-5G5; KangChen Biotech, Shanghai, China; dilution, 1:10,000) at 4°C overnight. The membranes were then incubated for 1 h at room temperature with the appropriate horseradish peroxidase conjugated-secondary antibodies. The blots were visualized using an enhanced chemiluminescence solution (EMD Millipore) and were exposed to X-ray film (Kodak, Tokyo, Japan). The density of the protein bands was analyzed using ImageJ 1.41 software (National Institutes of Health, Bethesda, MD, USA). The expression levels of the target proteins were normalized to those of GAPDH.

Protein extracts were resolved on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% freshly prepared milk-tris buffered saline Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with antibodies specific for β-actin (Abcam, Cambridge, MA)，HO-1, Bax, Bcl-2 (Beyotime), ERK1/2 and pERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Signals were detected with horseradish peroxidase–labelled secondary antibodies by chemiluminescence labelling.

Protein extraction and western blotting were performed as described previously.^{17 (link)}
Briefly, proteins were isolated by RIPA lysis buffer (Beyotime, China).
Then 24 μg protein was loaded and separated by 12% SDS-polyacrylamide gel
electrophoresis and transferred onto a polyvinylidene difluoride membrane and
incubated with antibodies: bax (#2774), bcl-2 (#3498), cleaved caspase-3
(#9661), cleaved caspase-8 (#8592), cleaved-PARP (#5625), p21 (#2947), p27
(#3686s), CDK1 (#4539), E-cadherin (#14472), PI3K (#17366), AKT (#4691), Erk
(#4370), JNK1 (#4668), mTOR (#2983), and GAPDH (#51332). These antibodies were
purchased from Cell Signaling Technology. MMP2 (sc-13595) and MMP9 (sc-21736)
were purchased from Santa and MCP-1 (66272-1-lg) was purchased from Proteintech.
The chemiluminescence signals were detected with an Amersham Imager 600 (GE,
USA).