Figure 1.
VEGF promotes FAK phosphorylation and translocation in endothelial cells. (A) Lysates of VEGF-stimulated primary HUVECs (20 ng/ml; 5 min), mouse brain and lung brain (2 μg/animal, 5 min) were prepared as described in Materials and methods and subjected to immunoblotting with anti-phosphotyrosine antibodies specific for aa 397, 407, 576, 577, 861, or 925 within FAK. The sensitivity and specificity of the phosphospecific antibodies were characterized in various tissues as described in the Materials and methods. These immunoblots are representative of three different experiments. (B) Translocation of endogenous FAK in VEGF-stimulated HUVECs (20 ng/ml; 5–60 min) to focal adhesions was determined by indirect immunofluorescence with an anti-FAK antibody in representative micrographs, as described in Materials and methods. Bar, 5 μm. (C) Representative FAK activity in lysates of VEGF-stimulated HUVECs (20 ng/ml; 5–60 min) was measured by immune complex in triplicate in vitro kinase assays as described in Materials and methods (P < 0.05). (D) Lysates of VEGF-stimulated HUVECs (20 ng/ml; 2–60 min) were subjected to immunoblotting with an anti-phosphotyrosine antibody specific for aa 397, 861, an anti-phospho Erk antibody, or an anti-FAK antibody. Each of these panels are representative of triplicate experiments.