IQGAP1 knockdown in endothelial cells reduces monocyte TEM. (A) HUVECs were fixed, permeabilized, stained for IQGAP1 and PECAM, and visualized using widefield fluorescence microscopy. Merged panel shows IQGAP1 in green and PECAM in red. Yellow indicates colocalization. Scale bar represents 50 µm. (B) HUVECs were transduced with the indicated shRNAs against IQGAP1 or control shRNA (Cont; scrambled). 3 d after the transduction, the cells were lysed and their protein content resolved using SDS-PAGE and probed using Western blotting. Actin is shown as a loading control. MW, molecular weight. (C) HUVECs treated as in B. visualized using immunofluorescence as in A. Both images were collected under the same settings in the same experiment and processed identically. Scale bar represents 50 µm. (D) The transmigration of monocytes across HUVECs grown on collagen gels transduced with IQGAP1 shRNAs or control as detailed in B. The mouse anti-human PECAM antibody hec7 was included as a control. Data represent the average and standard deviation of three independent experiments, each of which was comprised of at least three samples and >100 leukocytes scored per sample for each condition. *, P < 0.01.