Anti mmp 3

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).

Alantolactone (HY-N0038) was obtained from MedChem Express (MCE). Recombinant mouse IL-1β cytokine was provided from R&D Systems (501-RL-010, United States). Solarbio supplied safranin O liquor (Beijing, China). Primary antibodies applied in this research were: anti-iNOS, anti-MMP-13 which were acquired from Abcam (Shanghai, China), anti-COX2, anti-LC3Ⅱ/Ⅰ, anti-P-STAT3, anti-P-P65, anti-P-IκB, anti-P/T-mTOR which were purchased from CST (Beverly, MA, United States), anti-STAT3, anti-MMP-1, anti-MMP-3, anti-ATG5, anti-P62, anti-P65, anti-IκB, anti-P/T-PI3K, anti-P/T-AKT, anti-GAPDH which were afforded from Proteintech Group (Wuhan, Hubei, China) and anti-ADAMTS5 which was supplied from Boster Biological Technology (Wuhan, Hubei, China). Secondary antibodies, Cy3 and FITC Conjugated AffiniPure Goat Anti-Rabbit IgG, DAPI staining solution, collagenase type II and trypsin were got from Boster Biological Technology (Wuhan, Hubei, China). HanBio Inc. (Shanghai, China) served the mRFP-GFP-LC3 adenoviral vectors.

Brain tissues in the peri-ischemic area and primary astrocyte were used for western blot analysis. The protein concentration was measured using a BCA protein kit (Epizyme). The proteins (30 μg/group) were separated into a 5-10% gradient SDS-PAGE gel and then transferred onto PVDF membranes. After blocking with 5% milk, the bands were incubated overnight at 4°Cwith primary antibodies, including anti-CD63 (1:1000, #sc-15363, Santa Cruz Biotechnology), anti-TSG101 (1:1000, #ab83, Abcam), anti-TNF (1:500, #sc-52746, Santa Cruz Biotechnology), anti-MMP3 (1:1000, # 17873-1-AP, ProteinTech Group), anti-NFκB p65 (1:1000, #8242T, Cell Signaling Technology), or anti-β-actin (1:1000, #66009, ProteinTech Group). Then the membranes were incubated with HRP-conjugated secondary antibody (1:5000, #HA1006 or #HA1001, HUABIO) at room temperature for 1 h. Visualization analysis was performed using an enhanced chemiluminescence substrate (#SQ201, Epizyme) and an imaging system (Bio-Rad, Hercules, CA).