IQGAP1 recruits RacGAP1 to the tips of invasive pseudopods as cells migrate in 3D. (A and B) A2780 cells were subjected to control or IQGAP1 #1 RNAi and seeded onto CDMs. Cells were stimulated with cRGDfV as indicated for 2 h before fixing and staining with rabbit anti-RacGAP1/anti–rabbit Cy2 antibodies and phalloidin–Texas red. (C) A2780 cells stably expressing GFP, FLAG-RacGAP1^WT, FLAG-RacGAP1^249A, or FLAG-RacGAP1^249D on CDMs were stimulated with cRGDfV as indicated and fixed and stained with rabbit anti-IQGAP1/anti–rabbit Cy2 and mouse anti-FLAG/anti–mouse Cy3 antibodies. Images were captured using a spinning-disk confocal microscope, and representative pseudocolored images are shown. Zoomed insets correspond to areas indicated by dotted ROIs. Bars, 10 µm. Yellow arrows indicate direction of migration.