NRP1 knockdown reduces HGF-induced c-met phosphorylation and VEGF-A165– and HGF-induced signaling. (A) HUVECs were transiently transfected with 50 nM NRP1 siRNA-A, -B, or -C, or with 50 nM control siRNA-A, -B, or -C. NRP1 protein level was evaluated by Western blotting. (B) HUVECs were transfected with 50 nM NRP1 or control siRNA-A, deprived of serum, and incubated with or without 10 ng/mL HGF for 10 minutes. 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) HUVECs were transfected with 50 nM NRP1 or control siRNA-A, -B, or -C, deprived of serum, and incubated with or without HGF (2 or 10 ng/mL), or with VEGF-A165 (10 ng/mL). Cell lysates were analyzed by Western blotting with anti–phospho-ERK1/2 and anti–total ERK2 antibody (top; shown for siRNA-C). Bands were quantified by scanning densitometry, and results were normalized according to total ERK2 content (bottom). Data are expressed as a percentage of the maximal phosphorylation obtained in HUVEC transfected with control siRNAs. Values are means plus or minus SD of 3 independent experiments. **P < .01 (Student t test).
