FIGURE 1.

Time course of tunicamycin inhibition of VEGF-stimulated capillary endothelial cell proliferation. A, synchronized culture of capillary endothelial cells was treated with VEGF₁₆₅ (10 ng) in the presence or absence of tunicamycin (1 μg/ml), and cell numbers were counted microscopically. VEGF₁₆₅ was added prior to the addition of tunicamycin. B, phosphotyrosine kinase activity in capillary endothelial cells. The experiment was performed per instructions from the manufacturer, and measurements were made in an automated microplate reader/EIA plate reader (Model 2550, Bio-Rad) at 450 nm. The synchronized cells were treated with tunicamycin (1 μg/ml) for 3 h and then treated with VEGF₁₆₅ for 10 min. CBO-II was added 30 min prior to the addition of VEGF₁₆₅. C, status of VEGFR1 and VEGFR2 receptors. Synchronized cells were incubated with tunicamycin for 3 h-32 h, and the levels of total VEGFR1, phospho-VEGFR1, total VEGFR2, and phospho-VEGFR2 receptors were analyzed by Western blot using anti-VEGFR1 total (1:2,000; v/v), anti-phospho-VEGFR1 (1:1,000; v/v), anti-VEGFR2 total (1:2,000; v/v), and anti-phospho-VEGFR2 (1:1,000; v/v) antibodies. Actin (1:5,000; v/v) was used as a loading control. D, quantification of VEGFR1 and VEGFR2 receptors: Densitometer scanning (arbitrary unit) of the Western blots was plotted against the time of treatment. The results are an average from three representative immunoblots for each experiment.