IQGAP1 is required for the targeted recycling of the LBRC. (A) HUVECs grown on collagen gels and transduced with control or shRNA against IQGAP1 (knockdown, KD) as in Fig. 1 were subjected to the targeted recycling assay (described in Materials and methods), which follows the directed movement of LRBC to the site of transmigration. When visualized using immunofluorescence microscopy, recycled LBRC is observed as increased fluorescence adjacent to monocytes (visualized with anti-CD18). Staining of the same monolayers for VE-cadherin shows the expected gap in fluorescence where TEM is proceeding (denoted by the arrows). Images shown were contrast adjusted identically and are representative of three independent experiments. (B) Quantification of the monocyte position and LBRC recruitment shown in A. Monocytes were considered to be on the endothelial cell (EC) junction if they overlapped at all with the junction. Data shown are the average and standard deviation of three independent experiments, with at least 50 monocytes scored per experiment. Data from each experiment were normalized to the total adherent cells to account for subtle differences in adhesion on each monolayer. Scale bar represents 20 µm. Note that this is a snapshot of a short time point, so only approximately one third of adherent monocytes on control monolayers are actively transmigrating at this time. *, P < 0.01.