Figure 4.

NRP1 loss impairs VEGF165-induced SFK activation in human ECs. (A and B) Immunostaining of confluent HDMEC cultures in growth medium under nonpermeabilizing conditions with an antibody specific for human NRP1 (A) or under permeabilizing conditions with antibodies for CDH5 and NRP1 together with DAPI to visualize cell nuclei (B); three independent experiments. Single channels in B are shown separately in grayscale, and the boxed area is shown in higher magnification on the right. Bar, 50 µm. (C) Immunostaining of confluent HDMEC cultures under permeabilizing conditions for pSFK together with CDH5 and DAPI after serum withdrawal, followed by stimulation with VEGF165 for the indicated times (three independent experiments). The corresponding single pSFK channels are shown beneath each panel in grayscale as well as the rainbow pixel intensity scale. Bar, 50 µm. (D–F) Confluent HDMEC cultures transfected with si-control or siNRP1 were serum-starved and treated with VEGF165 for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (D), followed by quantification of pVEGFR2 (Y1175; E) and pSFK (F) induction relative to tVEGFR2 and GAPDH, respectively. Each of the two vertical lines indicates a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. Data for si-control and siNRP1-treated cells are expressed as ratio (E) or fold change, for VEGF165 treatment at different time points relative to 0 min (F) mean ± SEM; n = 4 independent experiments; asterisks indicate significant P-values for pSFK induction after VEGF165 treatment (*, P < 0.05; **, P < 0.01; paired Student’s t test). Hash tags indicate significant P-values for reduced pVEGFR2 and pSFK levels in siNRP1 versus si-control at the corresponding time points (^##, P < 0.01; unpaired Student’s t test).