NRP1 transduces ECM signals independently of VEGF165 and VEGFR2. (A–C) For phosphoproteomic screening, HDMECs were transfected with control or NRP1 siRNA before immunoblotting to confirm NRP1 knockdown (A) or stimulation with VEGF165 for 10 min (B) or FN for 30 min (C), followed by phosphokinase-antibody array screening. Phosphorylation of the indicated proteins in si-NRP1 relative to si-control transfected cells from 2 independent experiments, performed in duplicate each, were shown as mean fold change ± SEM. (D and E) To validate NRP1-regulated phosphoproteins identified in the phosphoproteomic screen, HDMECs were transiently transfected with control, NRP1 or VEGFR2 siRNA and stimulated with VEGF165 (D) or plated on FN (E) for the indicated times. Lysates were immunoblotted for the indicated proteins, with GAPDH serving as a loading control. Immunoblots are representative of 3 independent experiments. (F) pERK (T202/Y204) levels were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SEM. *, P < 0.05, Student’s t test.