Vegfa 66 828 1 ig

HUVECs and BMECs were lysed using radioimmunoprecipitation assay buffer (Biosharp, Hefei, China). To determine protein concentration, a Bicinchoninic Acid Protein Assay Kit (Biosharp) was utilized. Equivalent protein lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Then they were then subjected to western blot analysis. The primary antibodies against FOXO1 (2880S), cleaved caspase-3 (9661S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies anti-cyclin D1 (60186-1-1), anti-cyclin E1 (11554-1-AP), anti-Bim (22037-1-AP), anti-Bcl2 (60178-1-Ig), anti-Bax (60267-1-Ig), anti-matrix metalloproteinase (MMP)2 (66366-1-Ig), anti-MMP9 (N-terminal, 10375-2-AP), and anti-vascular endothelial growth factor A (VEGFA; 66828-1-Ig) were obtained from Proteintech. In addition, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG H&L (ZJ2020-M and ZJ2020-R, respectively; Bioworld) was applied as the secondary antibody. The protein bands were visualized via an electrochemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA).