Ab133425

After excessive anesthesia in rats leading to death, the tissue of wound was collected. Total protein was extracted, and after the determination of its concentration by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA), the protein was treated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation as well as transferred onto a nitrocellulose membrane. Src (ab133283, ABCAM, USA,1 : 2000), VE-cadherin (ab231227, ABCAM, USA,1 : 5000), Ang1 (ab133425, ABCAM, USA,1 : 5000), Tie-2 (19157-1-AP, Proteintech, USA,1 : 5000), and β-actin (ab8226, ABCAM, USA,1 : 5000) antibodies were added for incubation overnight at 4 °C. Then came the indoor cultivation (1 h) with the corresponding II antibodies. Enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) was carried out, and Fluor Chem 2.0 image analyzer was applied to analyze.

Western blot analysis was used to the determine the Ang-1 protein expression level in the infarcted myocardium (which was normalized to the protein levels of β-actin) 2 weeks following gene transfection. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene fluoride membranes and incubated with goat anti-Ang-1 antibody (ab133425; Abcam, Cambridge, UK) at 4°C overnight. The membranes were blocked with 5% non-fat milk and incubated with horseradish peroxidase-coupled mouse anti-goat IgG secondary antibodies (ab6789; Abcam) for 1 h at room temperature. The membranes were washed and exposed to X-ray film to detect the expression bands.

The myocardium (100 mg) was homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Proteins were separated by electrophoresis on an SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked in 5% nonfat milk and incubated with anti-Ang-1 antibody (1 : 1000; ab133425; Abcam, Cambridge, MA, USA) overnight at 4°C; then it was incubated with the HRP-conjugated IgG (1 : 2000; ab97057; Abcam, Cambridge, MA, USA) for 1 h at room temperature. The protein band was evaluated using the ECL chemiluminescence system (KeyuShenlan Scientific Co., Ltd., Beijing, China). The housekeeping protein (GAPDH) was used as the internal standard for the Ang-1 calculation. The ratio of protein/GAPDH was used to quantify the Ang-1 protein levels in each group.