Loss of cortactin leads to decreased levels of active Rap1, and Epac activation fully compensates the effect on permeability caused by down-regulation of cortactin. (A) Western blot analysis of pull-down assays for active Rap1 in HUVECs transfected with either control (ctrl) or cortactin-specific siRNA and for primary MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice. Total Rap1 levels were controlled by blotting aliquots of the whole cell lysates from which the pull-downs were performed. (B) Rap1 activation assays as in (A) performed with HUVECs transfected with either control or cortactin-specific siRNA or of MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice and additionally treated with 100 µM 8-pCPT-2Me-cAMP (+007) or vehicle (−007). (A and B) The depicted immunoblots are representative of three similar experiments. (C) Permeability for 250-kD FITC-dextran was analyzed in HUVECs transfected with either control or cortactin-specific siRNA and treated with either 100 µM 8-pCPT-2Me-cAMP (007) or vehicle. (D) Permeability for 250-kD FITC-dextran was analyzed in MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice as described for HUVECs in C. (C and D) Graphs are representative of three independent experiments. All data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.