Integrin trafficking suppresses Rac activity and activates RhoA through the RacGAP1–IQGAP1 complex. (A) A2780 cells were subjected to control or RacGAP1 oligo #6 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C; n ≥ 15/condition). (B) A2780 cells were subjected to control or IQGAP1 oligo #1 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto the CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C; n ≥ 8/condition). (C) A2780 cells stably expressing RacGAP1^WT, RacGAP1^249A, or RacGAP1^249D were transfected with Raichu-Rac and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C). Representative images are shown (n ≥ 8/condition). (D) A2780 cells stably expressing RacGAP1^WT, RacGAP1^249A, or RacGAP1^249D were transfected with Raichu-RhoA and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C). Representative images are shown (n ≥ 4/condition). Zoomed insets correspond to areas indicated by dotted ROIs. Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P < 0.5; ***, P < 0.001. Bars, 10 µm.