Anti timp3

Apatinib mesylate tablets (AITAN®; 425 mg/tablet) were purchased from Hengrui Medicine Company (Jiangsu, China). Tablets were dissolved in 100% dimethyl sulfoxide (DMSO; MP Biomedicals, Santa Ana, CA, USA) to a concentration of 4 mmol/L and stored at –20°C. The stock solution was diluted to a working concentration with DMEM (Gibco, Grand Island, NY, USA), and the final DMSO concentration in cell culture was less than 0.1%. The BCA kit was purchased from Google Biotechnology Ltd. (Wuhan, China). The RNA extraction reagent TRIzol and the PCR reagent Power SYBR® Green PCR Master Mix were purchased from Life Technologies (Grand Island, NY, USA), and the PCR primers were synthesized by Google Biotechnology Ltd. (Wuhan, China). Anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-7, anti-MMP-9, anti-TIMP-1, anti-TIMP-2, and anti-TIMP4 antibodies were purchased from Proteintech Group (Chicago, USA), and Anti-MMP-10, Anti-MMP-11, and Anti-MMP-16 antibodies were purchased from Biorbyt Ltd. (Cambridge, UK). Anti-TIMP-3 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). An NF-κB pathway sampler kit (9936T) was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

The Immunohistochemistry staining were performed with EnVisionTM System (DAKO, Carpinteria, CA, USA), and the slides were de-paraffinized, rehydrated, followed by 5 min antigen retrieval, 10 minutes of endogenous enzyme block, and incubated with primary antibody (anti-TIMP2 and anti-TIMP3, Santa Cruz) overnight at 4°C. Then the slides were incubated with EnVision-HRP secondary antibody for 1 hour and the signal were detected by diaminobenzidine (DAB), followed by hematoxylin counterstaining. Staining signals were photographed using an Olympus BX51 Microscope (Olympus, Tokyo, Japan).