Vegf b

The protein concentration was determined using the BCA assay. One hundred micrograms of total protein was loaded into each well and seperated by electrophoresis in a 10% SDS-PAGE gel. The proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.5% Tween-20 (PBST), the PVDF membranes were incubated overnight at 4˚C with SPARC (1:500; Cell Signaling Technology, Danvers, MA, USA), VEGF-A (1:500; Abcam, Cambridge, MA, USA), VEGF-B (1:500; Abcam), VEGF-C (1:500; Abcam), VEGF-D (1:500; Abcam), VEGF-E (1:500; Abcam) and GAPDH (1:1,000; Abcam) primary monoclonal antibodies in PBST buffer containing 0.1% Tween-20. The next day, the membranes were washed and incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) (1:2,000; Abcam). The intensity of protein staining was determined with ImageJ software.

Western blot was carried out with rabbit polyclonal antibody against VEGF-B (1:1000; Abcam), Sp1, (1:1000; Santa Cruz), rabbit-anti-rat VEGF-A (1:1000; Abcam), p-Sp1 (1:1000; Abcam), Akt and pAkt (1:1000; Cell Signaling). Rat left ventricles were removed and grinded in liquid nitrogen. The samples were collected and homogenized on ice in a 0.1% Tween-20 homogenization buffer containing protease inhibitors. 50 µg of proteins were resolved in 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). After being blocked with 5% nonfat milk, the membrane was incubated with primary antibody (1:1000 dilution) for 90 min followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, anti-mouse IgG, 1:10000, Jackson ImmunoResearch). Protein expression was visualized by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech) and quantified by densitometry [20] .