Figure 3.

NRP1 (neuropilin-1) cleavage in endothelial cells is mediated by ADAMs (a disintegrin and metalloproteinases) 9 and 10. Human umbilical vein endothelial cells (HUVECs) were transfected with control siRNA (siScr) or siRNAs specific for ADAM9 (A) or ADAM10 (B), and after 72 h were then treated with 25 ng/mL VEGF (vascular endothelial growth factor) for 60 min; cell lysates were then immunoblotted with the antibodies indicated. C, HUVECs were treated for 24 h with either no additions, or with DMSO (vehicle), or with the indicated concentrations of GI254023X, a specific inhibitor of ADAM10, and cell lysates were then immunoblotted with the antibodies indicated; quantification of the 10 kDa NRP1 cytoplasmic domain band is shown, *P<0.05 vs DMSO treatment, n≥3. D, Effects of single and double knockdown of ADAM9 and ADAM10 on the expression of NRP1 cytoplasmic domain fragments. The blots shown are representative of 4 different experiments. E, Quantification of the 10 kDa NRP1 cytoplasmic fragment from experiments in D, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, after knockdown of ADAMs 9 or 10 using single siRNAs at 200 nmol/L (black symbols), or double knockdown of ADAM9 plus ADAM10 (unfilled symbols) using combinations of siRNAs (total siRNA concentration 400 nmol/L); *P<0.05 vs Scr siRNA (200 nm) or Scr siRNA (400 nm) as appropriate. Values are presented as a scatterplot. Differences between samples were analyzed using 1-way ANOVA with the Bonferroni correction for multiple pairwise comparisons after testing for normality and equal variance using the Shapiro-Wilk and Levene tests, respectively.