HUVEC cells were a gift from Professor Yang Zhi-Hua (Department of Cell and Belinostat mouse Molecular Biology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) and were cultured in M200 basal culture media supplemented with low serum growth supplement (Cascade Biologics, PL, USA), 100 U/ml penicillin and 100 mg/ml streptomycin [23–25]. All cells were cultured at 37°C in a 5% CO2 humidified atmosphere. Flow cytometric assay Cells were collected, washed twice with phosphate buffered saline (PBS),
adjusted to 1 × 106 cells/ml, and incubated with ATP synthase subunit beta monoclonal antibody (1:300; MitoScience MS503, EA, USA) for 30 min at 4°C. After washing three times with PBS, fluorescein-isothiocyanate (FITC)-labeled Semaxanib research buy goat anti-mouse IgG (Jackson,WG, PA) diluted in PBS was added, incubated for 20 min at 4°C, then cells were washed three times with PBS, 1 ug/ml PI(Propidium Iodide, Sigma, St. Louis, MO, USA)) was added to exclude the dead cells and membrane
antigen expression was analyzed using a fluorescence-activated cell sorter (ESP Elite, Beckman Coulter, Fullerton, CA, USA). All experiments were performed three times. Production of functional F1F0 ATPase Î² subunit antibody Six to eight weeks old female BALB/c mice were subcutaneously immunized with hATP5B (F1F0 ATPase β subunit) which had been expressed using a prokaryotic selleck chemical system, as previously described , and mixed with Freund’s complete adjuvant (Sigma, St. Louis, MO, USA). The antibody valences in peripheral blood were determined using an ELISA as Gou, L. T. described , and three days after the last boost, Edoxaban 5 × 108 sensitized spleen cells were harvested, mixed and fused with 1 × 108 SP2/0 myeloma cells, in 50% polyethylene
glycol 1500 in a proportion of 8:1. The fused cells were plated in 96-well plates (6 × 105/well) and cultured for two weeks in RPMI 1640 with 10% fetal calf serum containing hypoxanthine, aminopterin, and thymidine to select for positive hybrid cells. The positive hybridoma cells were subcloned by limiting dilution, and 10–12 week old female BALB/c mice were inoculated with 3 × 106 hybridoma cells [3, 26]. The antibodies were further purified from the ascites via Protein-A affinity chromatography . The antibody with the highest valence against the F1F0 ATPase β subunit was named as McAb7E10 and used in further experiments. Western blotting and BIAcore analysis Cellular proteins were extracted in 40 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100 and supplemented with a cocktail of protease inhibitors. Equal amounts of protein were resolved on 12% SDS-PAGE gels then transferred to a PVDF membrane. After blocking with 5% non-fat milk, the membranes were incubated with McAb7E10 antibody overnight at 4°C, then with HRP-conjugated sheep anti-mouse IgG secondary antibody (Vector, Burlingame, CA, USA).