Figure 4: Western-blot analyses of phosphorylation of VEGFR-2, FAK, and Akt, by SDS-PAGE. Endothelial cells cultured on VEGFand FN-bound substrates were starved for 24 hours before the indicated times, and then lysed and supernatants were obtained. Equal amounts of protein were loaded in the lanes. Immunoblotting was performed with anti-phospho–VEGFR-2 antibody, anti-phospho–FAK antibody, and anti-phospho-Akt antibody as well as antibodies specific for the corresponding un-phosphorylated proteins. One day before the measurement of phosphorylation, culture medium was replaced with serum- and growth factor–free EBM-2 medium