Anti mmp 7

TBMS1, bought from National Institute for the control of Pharmaceutical and Biological Products (Beijing, China) with purity >98% by high-performance liquid chromatography (HPLC), was dissolved in DMSO to get a stock solution of 20 mmol/L and stored at −20°C. The stock solution was subsequently diluted to the desired concentration by a 1:1 mixture of DMEM/F12 medium when used (concentration of DMSO <1%). Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s nutrient mixture F12, fetal bovine serum (FBS), paraformaldehyde and agarose were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Propidium iodide (PI) was bought from BD Biosciences (San Jose, CA, USA). Phosphatase inhibitor, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5′-bromo-2′-deoxyuridine (BrdU), 4′,6-diamidino-2-phen-ylindole (DAPI) and polyvinylidene difluoride (PVDF) membrane were purchased from EMD Millipore (Billerica, MA, USA). Mouse monoclonal anti-c-Myc and anti-GAPDH were obtained from Abcam (Cambridge, UK). Rabbit monoclonal anti-PARP, anti-cleaved PARP (c-PARP), anti-caspase-3, anti-caspase-7, anti-caspase-8, anti-cleaved caspase-3 (c-caspase-3), anti-cleaved caspase-9 (c-caspase-9), anti-Bcl-2, anti-ERK1/2, anti-p-ERK1/2 and anti-MMP-7 were purchased from Cell Signaling Technology (Danvers, MA, USA). All antibodies were diluted according to the manufacturer’s instructions.

Immunoblotting was performed to detect the protein expression in osteosarcoma cell lines. Cultured or transfected cells were lysed in RIPA buffer with 1% PMSF. Protein was loaded onto a SDS-PAGE minigel and transferred onto a PVDF membrane. After being probed with primary antibodies [anti-Sirt1, anti-c-PARP, anti-Bak, and anti-Bcl-xl (Sigma-Aldrich); anti-MMP7, anti-Wnt5a, anti-β-catenin, and anti-pGSK3β (Cell Signaling Technology, Danvers, MA, USA); and anti-β-actin (Abcam, Cambridge, UK)] at 4°C overnight, the blots were subsequently incubated with HRP-conjugated secondary antibody (1:5,000). Signals were visualized using ECL Substrates (Millipore, Boston, MA, USA). β-Actin was used as an endogenous protein for normalization.