The actin-binding domain and IQ motifs of IQGAP1 are required for IQGAP1’s function in TEM. (A) iHUVECs grown on collagen gels, transduced with the indicated control, knockdown (KD), and reexpression constructs, and stimulated with TNFα were incubated with PBMCs for 1 h, after which the samples were washed, fixed, stained, and visualized using bright-field microscopy as described in Materials and methods. Data shown represent the percentage of monocytes that were found beneath the endothelial cell (EC) monolayer and are the average and standard deviation from three experiments, each of which comprised triplicate samples for each condition. *, P < 0.01 compared with the knockdown-FL rescue control. (B) The same experiment as in part A was performed using purified human neutrophils. Transmigration was allowed to proceed for 15 min. (C) iHUVEC grown on collagen gels, transduced, and stimulated as described above. 2 d after the transduction of the truncation constructs, the monolayers were treated with rhodamine-dextran (molecular weight, 10 kD) for 1 h. After four quick washes, the fluorescence that had passed through the monolayer into the collagen gel was quantified using a fluorescence plate reader. Data were collected for each sample and normalized to the average observed with the GFP control shRNA samples within each experiment. Data shown for A–C are the average and standard deviation from three independent experiments, each of which contains triplicate samples for each condition. None of the conditions were statistically different (P < 0.05) from any other. (D) The Miles assay, measuring the vascular leakage of Evans Blue, was performed on the ears and dorsal skin of WT and IQGAP1 knockout mice. No statistical difference was observed between the two groups. Data shown are for four mice for each group.