Mouse anti rat gapdh monoclonal antibody

Total protein of NSCs was extracted using a protein extraction kit and quantified using a BCA kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacture’s instruction. Equal amounts of total protein (20 μg) were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was blocked in TBS buffer with 5 % skim milk and 0.1 % Tween20 for 1 h, and incubated respectively with mouse anti-rat H3K27me3 monoclonal antibody (1:200 dilution, Abcam), mouse anti-rat GAPDH monoclonal antibody (1:1000 dilution, Abcam), mouse anti-rat ACAT2 monoclonal antibody (1:1000, Abcam), and mouse anti-rat β-tubulin monoclonal antibody (1:10000, Abcam) overnight at 4 °C. The membrane was then incubated with goat anti-mouse HRP-labeled secondary antibodies (1:2000 dilution, Abcam) at 37 °C for 2 h, and subjected to ECL detection for 5 min. The intensity of bands was detected by a Molecular Imager® ChemiDocTM XRS System (Bio-Rad Laboratories). The gray value of bands was analyzed by Image Lab 2.0 software (Bio-Rad Laboratories).

Anti-CD34 monoclonal antibody labeled by PE was purchased from Santa Cruz Co. USA (sc-7324). Anti-vWF antibody labeled by FITC, mouse anti-rat GAPDH monoclonal antibody, goat anti-mouse polyclonal IgG labeled by HRP and rabbit anti-rat MMP-9 monoclonal antibody were obtained from Abcam Co. USA (ab8822, ab9484, ab6789, ab76003). Hemolysin was purchased from BD Co. US (349202). ELISA kits for detecting the plasma level of VEGF, eNOS, NO, MMP-9 was purchased from Bejing Sizhengbo Biological Co. China (CRE0010). A total protein extraction kit was obtained from Applygen Technologies, Beijing, China. RIPA lysis solution was purchased from Beyotime Institute of Biotechnology (P0013B); ECL reagent was obtained from Millipore Co. (WBKLS0500). All other reagents were ultrapure grade.