Confluent HUVECs were washed two times with warm PBS before addition of either vehicle control ( 0.1% DMSO), VEGF (human recombinant VEGF165, R&D Systems,Abingdon, UK) or a mixture of VEGF and polyphenol (dp4 or EGCG). Treatments were for various times and concentrations and were performed using endothelial basal medium (endothelial cell growth medium-2 with no serum or
growth factors). After treatments, cells were lysed with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Protein content of lysates was determined
by a bicinchoninic acid assay (Sigma, Poole, UK).