Fig. 3.

Rap1 activation inhibits β-catenin reporter activity via KRIT1. (A) β-Catenin-dependent reporter activity in cells treated with 50 μM of OMe-cAMP, a membrane-permeant activator of Rap1, or that express constitutively active RapV12. TOPFlash activity is shown relative to the vector-transfected vehicle-treated (vec/veh) cells, n=3, P=0.0001 by ANOVA. *P<0.001 by Bonferroni post-hoc test compared with vector-transfected vehicle-treated cells; **P<0.001 compared with vehicle-treated, β-catenin expressing cells. (B) Rap1 inhibits β-catenin-dependent reporter activity induced by thrombin. TOPFlash activity is shown relative to vehicle-treated cells, n=3, P=0.001 by ANOVA. *P<0.01 by Bonferroni post-hoc test compared with vehicle-treated cells; **P<0.01 compared with thrombin-treated cells. (C) Activation of Rap1 does not inhibit reporter activity in KRIT1-depleted cells, n=3, P=0.0001 by ANOVA, *P<0.001 by Bonferroni post-hoc test compared with vector-transfected vehicle-treated cells. (D) Inhibition of nuclear β-catenin reporter activity requires the presence of cell-cell adhesion. Endothelial cells overexpressing β-catenin were plated at high (confluent) and low (sparse) cell densities on tissue culture-treated plastic, or sparsely in wells coated with an Fc-VE-cadherin extracellular domain fusion protein, in the presence or absence of 50 μM of OMe-cAMP. Control wells were blocked with excess non-immune mouse IgG and soluble cadherin-Fc in the presence of calcium to assess background binding. TOPFlash activity is normalized to vector-transfected vehicle-treated cells, n=3, P=0.001 by ANOVA, *P<0.01 by Bonferroni post-hoc test compared with vector-transfected vehicle-treated cells, **P<0.01 compared with β-catenin overexpressing cells.