Figure 5: Nrp1 influences Smad2/3 activation.

(a–c) Representative confocal images of wt sprouts from EBs immunolabelled for pSmad2. The outline of the wt sprouts is indicated with a dashed line using the endogenous DsRED marker. DAPI staining (not shown) was used to mark nuclei (blue line). Sprouts from (a) untreated EBs, (b) treated with 2 ng ml⁻¹ Tgf-β for 1 h and(c) treated with 10 μM SB-421543 for 4 h. Scale bar, 13 μm. (d,e) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml⁻¹ TGF-β for 1 h. Full western blots are shown in Supplementary Fig. 10. A representative blot of six is shown; P=0.0012 NRP1 siRNA compared with control. (f,g) Proteins from P4 HUVEC transfected with control-GFP and NRP1–GFP–His construct for 24 h, with or without stimulation with 2 ng ml⁻¹ TGF-β for 1 h were assessed for SMAD2 phosphorylation. Full western blots are shown in Supplementary Fig. 11. A representative blot of four is shown; P=0.0017 NRP1 overexpression compared with control. (e,g) Quantification of pSMAD2 protein normalized to SMAD2/3. (h–k) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml⁻¹ TGF-β for 1 h. Full western blots are shown in Supplementary Figs 12 and 13. (i) Quantification of pSMAD2 protein normalized to SMAD2/3; P=0.0848 Nrp1 siRNA compared with control. (k) Quantification of pSMAD3 protein normalized to SMAD3; P=0.0181 NRP1 siRNA compared with control. (l,m) Western blot analysis of P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 10 ng ml⁻¹ BMP9 for 15 and 30 min. Full western blots are shown in Supplementary Fig. 14. A representative blot of four is shown. (m) Quantification of pSMAD2/3 protein normalized to SMAD2; P<0.0011 NRP1 siRNA compared with control. All values represent mean±s.e.m. DAPI, 4,6-diamidino-2-phenylindole; NS, not significant.