Silencing or deficiency of cortactin enhances permeability of cultured endothelial cell layers. (A) HUVECs were transfected with either control (ctrl) or cortactin-specific siRNAs. 48 h later, cell lysates were immunoblotted for cortactin expression (top). Cortactin immunoblots of primary MLECs isolated from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice are shown. Equal loading was controlled by blotting for tubulin (bottom). Blots are representative of four independent experiments. (B) Micrographs of HUVEC monolayers transfected with control and cortactin siRNA and MLEC monolayers of Cttn^WT/WT (WT) and Cttn^del/del (KO) mice grown on transwell filters parallel to the experiment shown in C–E stained for VE-cadherin. Images are representative of four independent experiments. Bar, 20 µm. (C) Paracellular permeability for 250-kD FITC-dextran was determined for HUVECs transfected without oligonucleotides (set to 100%) or with either control or cortactin-specific siRNAs cultured on transwell filters (0.4-µm pore size). (D) Measurement of transendothelial electrical resistance (TER) of HUVECs treated as described in C. (E) Paracellular permeability of MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice for 250 kD (left) and 150 kD FITC-dextran (right). (C–E) All data are means ± SD. n = 6 for each group in three independent experiments. *, P < 0.05; **, P < 0.005.