Total protein was extracted from the rat’s thoracic aorta with a protein extraction kit (invent, SA-03-BV). Afterward, protein samples were separated by SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) polyvinylidene fluoride (PVDF) electrophoresis, and target protein bands in the gel were transferred to PVDF membrane. Subsequently, the PVDF membrane was blocked with 5% milk for 1 h, and was incubated with primary antibody (anti-eNOS, 1:1000, Abcam, ab199956, Cambridge, MA, USA; anti-AngII, 1:1000, Abcam, ab124734, Cambridge, MA, USA) overnight at 4°C. Finally, the PVDF membrane strip was incubated with horseradish peroxidase (HRP)-conjugated second antibody (Wuhan Sanying, SA00001-2) for 1 h, and was visualized by ChemiDoc™ Touch Gel imaging system (Bio-Rad, Hercules, CA, USA). The expression of target protein was measured using Image-Pro Plus 6.0 software,17 (link)
and was compared with GAPDH.
and was compared with GAPDH.
