Effects of Rac1 or myosin II inhibitors on Rac1 activity. (A) CHO-α4WT, CHO-α4Y991A, and CHO-α4S988A cells transfected with RFP-PAK-PBD were plated on VCAM-1 and imaged for GTP-loaded Rac1 (red) and GFP-tagged α4 integrin by dual-color confocal microscopy in the presence of vehicle control, NSC23766 (NSC) for CHO-α4Y991A, or blebbistatin for CHO-α4S988A. (B) Rac1 activity was quantified by measuring the RFP-PAK-PBD red fluorescence intensity for each cell type normalized by GFP-tagged α4 integrin. Data represent means ± SEM of ≥50 cells from two independent experiments. (C) The overall level of GTP-loaded Rac1 was quantified in the presence of vehicle control or indicated treatments using ELISA. Data represent means ± SEM from four independent experiments. *, P < 0.05. Ctrl, control; Bleb, blebbistatin.