ZO-1 is required for junctional recruitment of JACOP and p114RhoGEF. (A and B) HDMEC were transfected as indicated with siRNAs and were then analyzed by immunoblotting (A) or immunofluorescence (B). (C) HDMEC were extracted and subjected to immunoprecipitation with anti–ZO-1 antibodies. The precipitates were then analyzed by immunoblotting for ZO-1, JACOP, and vinculin. (D) The number of vinculin-positive focal adhesions per cell were counted in images such as those shown in B. Shown are means ± 1 SD (error bars); n = 15. (E) HDMEC were extracted and p114RhoGEF was immunoprecipitated. Precipitates were analyzed by immunoblotting for JACOP and vinculin. Bars, 30 µm.