(A) Decreased cell surface VE-cadherin in Ad-FGFR1DN–transduced BAECs. Quiescent BAECs were transduced with Ad-GFP or Ad-FGFR1DN and the monolayer was treated with biotin to label cell surface proteins. Biotinylated proteins were subjected to Western blot analysis and probed for VE-cadherin or N-cadherin. Total cell lysates from the same samples were also analyzed by Western blotting to evaluate total protein expression. (B) Western blot analysis of expression of junction proteins. Transduced BAECs were harvested at the indicated time points, and total cell lysates were analyzed by Western blotting. (C) Effect of FGF treatment on VE-cadherin interaction with catenins. BAEC monolayers were maintained in 0.5% FBS basal medium for 24 hours and then stimulated with FGF1 (50 ng/ml) for the indicated times. Total cell lysates were immunoprecipitated with anti–VE-cadherin antibody and blotted with p120-catenin, β-catenin, or VE-cadherin antibody. (D) FGFR signaling requirement for VE-cadherin interaction with catenins. Subconfluent BAEC monolayer (~70% confluency) was transduced with Ad-GFP or Ad-FGFR1DN, and cells were grown to form mature junctions. At indicated time points, total cell lysates were prepared and immunoprecipitated with VE-cadherin antibody followed by immunoblotting with p120-catenin, β-catenin, or VE-cadherin antibody. As the monolayer forms mature junctions, VE-cadherin/p120-catenin interaction increases (Ad-GFP), whereas this interaction was decreased in Ad-FGFR1DN–treated cells. Nontransduced cells were treated with 100 μM PV for 10 minutes prior to cell lysis, showing disrupted VE-cadherin/p120 interaction.