Fig. 6. Expression of endothelial cell markers in FGFR-1 ^-/- EBs. (A) Real-time PCR to determine CD31, VEGF-C, Ephrin B2 and VEGFR-2, VEGFR-1, VEGF-A transcription levels in 8-day-old FGFR^+/- (+/-) or FGFR^-/- (-/-) EBs cultured in basal conditions (i.e. without addition of exogenous growth factors). Changes are relative to ß-actin transcript levels. (B; right) Immunohistochemical staining for VEGFR-2 in FGFR-1^-/- EBs cultured for 8 days under basal condition. Scale bars: 200 µm. (Left) Expression of VEGFR-2 protein in total cell lysates of basal treated FGFR-1^+/- (+/-) and FGFR-1^-/- (-/-) 8-day-old EBs. ß-catenin was used as loading control. (C) Immunocomplex kinase assay of VEGFR-2 on day-8 FGFR-1^+/- (+/-) or FGFR-1^-/- (-/-) EBs cultured under basal conditions (without exogenous growth factors) and unstimulated (-) or stimulated (+) with 100 ng/ml of VEGF-A for 10 minutes. Slower migrating, kinase active VEGFR-2 appeared in the FGFR-1^-/- EBs (arrowhead). (D) CD31 staining of FGFR-1^-/- EBs in basal conditions treated with either control anti-rat IgG, neutralizing anti-VEGFR-2 (30 µg/ml), or anti-VEGF-A (5 µg/ml) antibodies from day 6-8. Scale bars: 200 µm. (E) Quantification of CD31 staining as area (i.e. area without holes) in 8-day FGFR-1^-/- EBs treated with anti-VEGFR-2 or anti-VEGF-A antibodies from day 6-8. Results are expressed as percentage of FGFR-1^-/- EBs treated with control IgG (mean±s.d., n=4). ^*P=0.002 for anti-VEGFR-2 treatment compared to control. ^*P=0.0306 for anti-VEGF-A treatment compared to control.