2.7 Western Blot
Total protein was extracted from cells in each group with strong RIPA lysate containing protease inhibitors. After ultrasonic treatment, protein lysates were centrifuged at 4℃and 12000rpm for 10min. Protein concentration was then and determined by the BCA method. Next, we added SDS-PAGE protein loading buffer to each sample and boiled the sample at 100℃ for 10 min to denature proteins. A 10% SDS-polyacrylamide gel was used to separate the protein samples which were then transferred to PVDF membranes. The membranes were then blocked with 7% skimmed milk at room temperature for 1h. Then, the membranes were incubated with anti-twist1 antibody (1:500), anti-angiopoietin1 antibody (1:1000), anti-angiopoietin2 antibody (1:1000), anti-tie2 antibody (1:1000) and a β-actin antibody (1:5000). Membranes were incubated overnight on a shaker in a 4℃ refrigerator. The following morning, the membranes were washed three times with PBST and then incubated with horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (H + L) or HRP-labeled goat anti-mouse IgG (H + L) at room temperature for 1 h. thereafter, the membranes were washed three times with PBST. ECL chemiluminescence was then used to identify positive antibody binding and Image J software was used to analyze the resultant protein bands.