Western Blot Analysis of Vascular Proteins

As described previously [26 (link)], tissues were homogenized on ice in a cell lysis buffer. The total protein of carotid blood vessels and the cells were extracted and the protein concentration was measured quantitatively using a BCA protein kit (AR1189, BOSTER). A quantity of 40 µg tissue protein or 10 μg cell protein were separated by 12.5% sodium dodecy1 sulfate polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked for 16 min using Ncmblot blocking buffer (P30500, New Cell & Molecular Biotech, Suzhou, China). PVDF was incubated with primary antibodies against anti-NOX2 (1:1000, dilution), anti-NOX4 (1:1000, dilution), anti-DRP1 (1:1000, dilution), anti-TRPM2 (1:300, dilution), anti-NHE1 (1:1000, dilution), and anti-GAPDH (1:10,000, dilution) and anti-VDAC (1:1000, dilution) at 4 °C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the protein bands were visualized by an ECL kit (P10100, Beyotime, Shanghai, China) on a ChemiDoc imaging system (Bio-Red, Hercules, CA, USA). The intensity (area X density) of the individual band on Western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). GAPDH and VDAC served as a loading control.