Enzyme-linked immunosorbent assay (ELISA)

Cells were transfected with pSV-VEGF or pNSE-VEGF and incubated under normoxic or hypoxic conditions. The supernatant sampling was performed each 2, 3, 5 and 7 days after transfection. Tissue samples were prepared same as luciferase assay protocol above. An ELISA kit (Abfrontier, Seoul, Korea) was used to measure the amounts of VEGF secreted from the cells in accordance with the manufacturer’s protocol. Samples were added to the wells of the pre-coated with VEGF monoclonal antibodies plate and incubated at 37 °C for 90-min. After the cell culture media samples were discarded, 0.1 ml of biotinylated VEGF antibody was added to each well and incubated at 37 °C for 60-min. Plates were washed three times in 0.01 M PBS. An Avidin–Biotin–Peroxidase Complex (ABC) working solution was added to each well and incubated at 37 °C for 30-min. The ABC solution was removed, wells were washed five times, and 90 μl of tetramethylbenzidine (TMB) color-developing agent was added and incubated at 25 °C in the dark for 20-min. After incubation, TMB stop solution was added, and catalyzed TMB had changed to yellow. The density of yellow was measured at an absorbance–wavelength of 450 nm. The relative amount of VEGF in each sample was determined with human VEGF standard curve.