Anti cox 2 antibody

Proteins were extracted from left ventricular myocardium with RiPA Buffer (Thermo Scientific) and protease inhibitor was added. Protein concentration was determined by BCA method (Solarbio, Beijing, China). Proteins were taken at 50 μg and denatured at 100°C for 5 mins. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic equipment (Bio-Rad, CA, USA). After being blocked with 3% BSA for 2 hours at room temperature, the membranes were incubated with anti-PI3K antibody, anti-phospho-PI3K antibody, anti-eNOS antibody, anti-phospho-eNOS (1 : 1000, Bioss, Beijing, China), anti-COX-2 antibody, anti-HMGB-1 antibody (1 : 1000, Proteintech, Chicago, IL, USA), and anti-ECE-1 antibody (1 : 2000, Signalway Antibody, College Park, MD, USA), primary antibodies overnight at 4°C. Next day, after being washed with TBST for 3 times, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1 : 25000, 1 h, Zhongshanjinqiao, Shanghai, China) at room temperature. After being washed with TBST for 3 times, the membranes were visualized by Pierce™ ECL Western Blotting Substrate and imaged using FluorChem E Imaging System (Protein Simple, USA), which were normalized to β-actin as an internal control.