VE-cadherin inhibits FGF signaling in ECs. (A) qRT-PCR analysis of Fgf-2 expression in VE and N cells. The means ± SD are graphed (n = 3; **P < .01). (B) Quantification of FGF-2 induced migration in VE and N cells. The means ± SD are graphed (n = 4; **P < .01; *P < .05). (C) Validation of expression levels of migration-related genes induced by FGF-2 in N and VE cells analyzed by qRT-PCR. The means ± SD are graphed (n = 3; **P < .01; *P < .05). (D) Immunoblots of p-FRS-2α and total FRS-2α from recently confluent (25-28 000 cell/cm2,49 ) N and VE cells, after 10 minutes of FGF-2 stimulation (20 ng/mL). Vinculin is used as loading control. (B-D) PD = PD173074, FGFr inhibitor, (2 hours prior stimulation, 200nM). (E) Coimmunoprecipitation of both FGFr1 and Dep-1 with VE-cadherin but not with N-cadherin in VE and N cells. (F) Quantification of the FGFr1 protein level in VE and N cells. Columns are means ± SEM (n = 3; *P < .05). (G) Immunoblots of phospho-FRS-2α and total FRS-2α from N and VE cells after Dep-1 knock-down and 10 minutes of FGF-2 stimulation (20 ng/mL). Vinculin is used as loading control. (H) Coimmunoprecipitation of N-cadherin and FGFr1 in cells overexpressing exogenous N-cadherin (N-overexpressing cells). (A-H) Cells have been cultured in starving medium for 18-20 hours before performing the experiments.
