IQGAP1 is recruited to anti-PECAM–coated beads. (A) HUVECs were incubated with PBMCs for 10 min before being fixed, stained for IQGAP1 and PECAM, and visualized using immunofluorescence microscopy. Deconvolved images shown are slices from a z-series of a monocyte in the act of TEM and show the monocyte (arrowhead in each slice) cell body above the monolayer, in the middle of a transmigration pore, and its pseudopod below the monolayer. Arrows point to endothelial cell borders. Scale bar represents 25 µm. Bottom panels show the yz orthogonal view along the dashed line displayed in the “Below” panel. The thin dotted line indicates the surface of the endothelium. (B) HUVECs were incubated with beads coated with nonspecific mouse IgG (mIgG) or mouse anti-human MHC (α-MHC), ICAM-1 (α-ICAM-1), or PECAM (α-PECAM) antibodies for 20 min and then fixed, stained for IQGAP1 (red) and junctions (anti-VE-cadherin, green), and visualized using differential interference contrast (left column) and confocal immunofluorescence (right column). Arrowheads denote beads displaying IQGAP1 enrichment. Scale bar represents 25 µm. (C) Quantitation of the data shown in B. A bead was considered to have enriched IQGAP1 if the adjacent fluorescence signal was at least 1.5-fold higher than the nearby off-bead signal. Data shown are the average and standard deviation from three experiments, with >100 beads quantified per experiment per condition. *, P < 0.05 relative to mIgG control. Representative images are shown.