Total soluble protein extracted from cells was resolved on 10% SDS polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. Blots were blocked with 5% milk followed by an overnight incubation with vascular endothelial growth factor (VEGF) antibody (R&D Systems, 1:300), HFG antibody (Abcam, Cambridge, MA, USA, 1:950), HIF-1α antibody (R&D Systems, 1:500), Ang-1 antibody (R&D Systems, 1:600), and GAPDH antibody (Beyotime, Beijing, China, 1:1000). The blots were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (Beyotime) and visualized using enhanced chemiluminescence substrate (Beyotime).
Total soluble proteins extracted from cells were resolved on 10% SDS polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes. After an overnight incubation with VEGF antibody (R&D Systems, 1:300), HFG antibody (Abcam, 1:950), HIF-1α antibody (R&D Systems, 1:500), Ang-1 antibody (R&D Systems, 1:600) and GAPDH antibody (Beyotime, Beijing, China, 1:1000), the blots were blocked with 5% milk, incubated with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (Beyotime), and finally visualized using enhanced chemiluminescence substrate (Beyotime).
Total soluble proteins extracted from cells were resolved on 10% SDS polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes. After an overnight incubation with VEGF antibody (R&D Systems, 1:300), HFG antibody (Abcam, 1:950), HIF-1α antibody (R&D Systems, 1:500), Ang-1 antibody (R&D Systems, 1:600) and GAPDH antibody (Beyotime, Beijing, China, 1:1000), the blots were blocked with 5% milk, incubated with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (Beyotime), and finally visualized using enhanced chemiluminescence substrate (Beyotime).