2.6. Western Blotting

The total protein of HUVEC was obtained by using a RIPA lysis buffer (Cell Signal Technology, Danvers, MA, USA) containing protease inhibitors. The proteins were then separated by SDS-PAGE (10%) with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (MilliPore, Bilerika, Ma, USA). After sealing with 5% skimmed milk and TBST, it was cultivated all night long at 4°C with anti-Bcl-2 (1 : 500), Bax (1 : 500), cle-caspase-3 (1 : 500), or GAPDH(1 : 1000) (Abcam, Cambridge, Massachusetts, USA). Then, at indoor temperature, it was immersed in horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1 : 1000; Wuhan Boster Biological Technology Co., Ltd.) for 1-hour culture, followed by 3 times of rinsing with PBS, 5 min each. In a dark room, development was carried out under the prerequisite that the excess liquid on the film was blotted with a filter paper and then illuminated by ECL to develop.