Transfection of RAC2[E62K] drives increased ROS production, increased RAC2[E62K] association with PAK-protein binding domain, increased pAKT, and increased membrane ruffling and macropinocytosis. (A) Diogenes assay to measure ROS production in COS-7 cells transfected with NADPH oxidase components (gp91^phox, p47^phox, p67^phox) and either RAC2[WT] (top), RAC2[E62K] (middle), or GFP (bottom) without stimulation (left) or after addition of 1 μM PMA (right). (B) Cumulative ROS production without (open bar) or with (filled bar) PMA stimulation; graph shows average ± standard error of the mean (SEM) of 1 representative experiment. (C) Immunoprecipitation of COS-7 cells transfected with RAC2[WT] or RAC2[E62K] using PAK1-PBD. Graph shows average ± SEM of 3 independent experiments. ***P = .0004. (D) Lysates from COS-7 cells transfected with RAC2[WT], RAC2[E62K], or untransfected were immunoblotted and stained for total AKT (tAKT) or phospho-AKT (S473). Bands were quantified by densitometry and the ratio of active, phospho-AKT/tAKT was plotted. Graph shows average ± SEM of 3 independent experiments (supplemental Figure 2B-D). (E) Confocal images of RAW264.7 cells (top, original magnification ×333) or COS-7 cells (bottom, original magnification ×235) transfected with RAC2[WT] (left) or RAC2[E62K] (right) and GFP as a transfection control. Cells were stained with Alexa-594-phalloidin (orange) to detect F-actin and Alexa-647-anti-RAC2 (red). Images were captured by a Zeiss LSM 880 confocal microscope and processed using the ZEN 2.3 lite program.