NRP1 and NRP2 double knockdown leads to effective inhibition of VEGF-A165– and HGF-mediated signaling. HUVECs were transfected with 50 nM NRP1 siRNA-C, NRP2 siRNA, or both, or with a control siRNA-C, deprived of serum, and incubated with or without 10 ng/mL of HGF, 10 ng/mL of VEGF-A165, or 50 ng/mL of IGF-I for 10 minutes. (A) NRP2 protein level was evaluated by Western blotting. (B) c-met was immunoprecipitated from cell lysates and subjected to Western blotting with an antiphosphotyrosine or anti–c-met antibody (left). The phosphoprotein content was estimated by scanning densitometry analysis and normalized according to total c-met levels (right). (C-E) Cell lysates were analyzed by Western blotting with anti–phospho-ERK1/2 and anti–total ERK2 antibody (left). Bands were quantified by scanning densitometry, and results were normalized according to total ERK2 content (right). Data are expressed as percentages of the maximal phosphorylation obtained in HUVECs transfected with control siRNA. Values are means plus or minus SD of 3 independent experiments. **P < .01; ***P < .001 (Student t test).
