ROS are required for AKT phosphorylation. (A) Wild-type neutrophils were pretreated with NAC (10mM) or Trolox (100μM) for 10 minutes after stimulation with 1μM fMLP for 30, 60, and 120 seconds. Phosphorylation of AKT (Thr-308) and total AKT were detected by immunoblot assay. (B) The ratio of phospho-AKT/total AKT was determined using densitometry, and the average of 3 independent experiments ± SD is shown. (C) Subcellular localization of a GFP-tagged PH domain of AKT was monitored in live neutrophils. Upon stimulation with fMLP (asterisk depicts the position of the microcapillary pipette tip), PH-AKT-GFP is recruited to the leading edge (panel 2). At t = 82 seconds (panel 3), NAC (10mM) was added to the incubation medium, inducing redistribution of PH-AKT-GFP. Using image analysis software, density profiles were generated and the relative accumulation at the front and back of the cells was quantified (last panel, n > 10 cells). (D-E) Phosphorylation of AKT (Thr-308) was quantified by immunoblot assay in gp91^−/− neutrophils (E) and control cells (C57/BL6; D). Cells were treated with 50μM H₂O₂, 10mM NAC, and 100μM Trolox, as indicated, and stimulated with 1μM fMLP for 1 minute. Bar diagrams represent the average of 3 independent experiments ± SD. *P < .05.