ROS signaling is required for uncapping activity. (A) Uncapping of actin filaments was measured by the fMLP-induced release of CapZ from partially permeabilized neutrophils. Cells were pretreated with Trolox or mock-treated for 15 minutes before stimulation with 1μM fMLP for 1 minute. Cells and supernatant were separated by centrifugation and analyzed by SDS-PAGE and immunoblotting for CapZ. (B) CapZ in pellets and supernatant were quantified by immunoblotting and subsequent densitometric analysis. Uncapping activity was quantified in 3 independent experiments by determining the ratio between CapZ in supernatant and pellet. The bar diagram depicts the averages ± SEM (n = 3). (C) CapZ-uncapping experiments were performed as described above using neutrophils from Gp91^−/− and C57/BL6 control mice. Cells were stimulated with fMLP (1μM) and H₂O₂ (50μM) and pretreated with Trolox (100μM) where indicated. Bar diagrams depict averages ± SD (n = 3). Asterisks indicate significance, P < .05. (D) Model describing the redox-mediated amplification loop. Activation of Rac induces ROS formation by NOX2, which oxidizes PTEN. The decrease in PTEN activity results in increased PtdIns(3,4,5)P₃ levels, facilitating guanine nucleotide exchange factor activity toward Rac. Subsequent Rac activity induces uncapping of actin filaments and local actin polymerization required for directional migration.