Blocking of VEGF signaling does not impair Notch signaling. (A) Regimen of antibody application. The neutralizing antibody against mouse VEGF (N-mVEGF) was injected into the tail vein on 2 consecutive days (E13.5 and E14.5), and embryos were analyzed at E15.5. (B) Analysis of VEGF-A and Notch activity. Staining for phosphorylated VEGFR2 and eNos as a downstream target of VEGFR2 confirms that VEGF-A signaling is effectively inhibited (a,b,e,f). VEGFR2 itself is still normally expressed (c,d). Notch activation (m,n), the expression of Notch ligands Dll1 and Dll4 (i,l) and of EphrinB2 (g,h) is not changed in N-mVEGF–treated embryos. Green indicates α-smooth muscle actin; red indicates antigen. a′ to n′ show magnifications. (C) Western blot analysis of markers analyzed in panel B. Actin was used as a loading control.