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.