The blocking anti-NRP1 and anti-NRP2 antibodies decreases VEGF-A₁₆₅– and HGF-induced cellular responses in HUVECs. Serum-deprived HUVEC were preincubated with 20 μg/mL anti-NRP1 IgG or nonimmune IgG and then stimulated for 10 minutes with HGF or VEGF-A₁₆₅. (A) ERK1/2 phosphorylation was analyzed by Western blotting. Figures (bottom) indicate phospho-ERK1/2 content as estimated by scanning densitometry with normalization according to ERK2 content. Data are expressed as a percentage of the maximal phosphorylation obtained in HUVECs stimulated with 10 ng/mL HGF in the presence of nonimmune IgG. (B) DNA synthesis was determined by [³H]thymidine incorporation. Data are expressed as fold stimulation of basal incorporation observed in unstimulated cells in the presence of nonimmune IgG. Values are means plus or minus SD of 3 independent experiments performed in triplicate. (C) Migration was analyzed by Transwell assay. CM-Dil–stained HUVECs were preincubated with 20 μg of anti-NRP1 IgG or nonimmune IgG and dispensed into the upper chamber of the Fluoroblok inserts. Migration was induced by adding 10 ng/mL of HGF or VEGF-A₁₆₅ to the lower chamber. The number of migrating cells was quantified by spectrofluorimetry. The results shown are means plus or minus SD of 3 independent experiments performed in triplicate. (D) HUVEC were preincubated with 20 μg/mL of anti-NRP1 IgG, anti-NRP2 IgG, or both, or with a nonimmune IgG and stimulated with HGF or VEGF-A₁₆₅. DNA synthesis was determined by [³H]thymidine incorporation. Data are expressed as fold stimulation of basal incorporation observed in unstimulated cells in the presence of non immune IgG. Values are means plus or minus SD of 3 independent experiments performed in triplicate. *P < .05; **P < .01; ***P < .001 (Student t test).