Total cellular protein was extracted with RIPA lysate buffer (Boster Biological Technology) containing protease inhibitor and phosphorylase inhibitor (Boster Biological Technology), and determined protein concentration with BCA protein detection kit (Boster Biological Technology), then separated by SDS-PAGE electrophoresis kit (cwbiotech). The amount of each group proteins was 20μg. After separation, the proteins were transferred to PVDF membrane (0.22um). When sealed by 0.5% non-fat milk powder still 1 h, the PVDF membrane was incubated with polyclonal primary antibody and secondary antibody combined with HRP. The membrane was colored by ECL enhanced chemiluminescence kit (Boster Biological Technology) and formed by Biological Spectrum Image System Scanning.
Primary antibodies used in this study include anti-β-actin(1:5000, AP0060, Bioworld Technology, Inc), anti-bcl-2(1:1000, AF6139, Affinity), anti-Bax(1:1000, AF0120, Affinity), anti-p-VEGFR-2(1:500, AF3279, Affinity), anti-MMP-2(1:500, AF5330, Affinity). The secondary antibodies include HRP-conjugated affinipure goat anti-Rabbit IgG(1:5000, Boster Biological Technology, BA1054).
Primary antibodies used in this study include anti-β-actin(1:5000, AP0060, Bioworld Technology, Inc), anti-bcl-2(1:1000, AF6139, Affinity), anti-Bax(1:1000, AF0120, Affinity), anti-p-VEGFR-2(1:500, AF3279, Affinity), anti-MMP-2(1:500, AF5330, Affinity). The secondary antibodies include HRP-conjugated affinipure goat anti-Rabbit IgG(1:5000, Boster Biological Technology, BA1054).