Western blot

Western blot analysis was performed to detect the expression of Bax and Caspase 3 in HUVECs treated by Ang II and Ang II + BBA. Cellular lysates from HUVECs in each group were first isolated by RIPA lysis buffer. Then, total proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 12% polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skim milk in TBST for 2 h, and specific diluted primary antibody (anti-Bax, anti-Caspase 3, Sigma, USA) was added. After incubation overnight at 4°C, the membrane was washed with TBST 3 times and incubated for another 2 h at 25°C with FITC-conjugated secondary antibody (Sigma, USA). Finally, the samples were washed with TBST, and semi-quantitative gel images were analyzed by use of an automatic gel imager using Quantity One software (Bio-Rad, USA). During this process, GAPHD was used as the internal control.