ROS production in SS RBC is mediated by a PKC–RacGTP signaling axis. Fraction 1 SS or AA RBC were preincubated with inhibitors or inducers for 1 hour prior to staining with CM-H2-DCFDA. (A) ROS levels in SS RBC are decreased by preincubation with the Rac-specific small-molecule inhibitor NSC23766 (NSC), indicating that Rac-GTP mediates ROS production in erythrocytes. (B,C) ROS levels in SS RBC are (B) increased by preincubation with the PKC activator PMA and (C) decreased by the PKC inhibitor calphostin (500 nM), indicating that PKC activity increases ROS production in erythrocytes. (D) ROS levels in SS RBC are decreased by preincubation with the cell-permeable calcium chelator BAPTA-AM (50 µM), indicating that free calcium, a key activator of classic PKC isoforms, is necessary for ROS generation in erythrocytes. (E) Fraction 1 SS RBC were incubated with 500 µM NSC23766 (NSC), 2 µM PMA (PMA), or both (NSC/PMA) for 1 hour before staining with CM-H2-DCFDA to detect ROS levels. Inhibition of Rac-GTP activity with NSC23766 blocks the PKC-mediated induction of ROS production in SS RBC, indicating that Rac-GTP acts downstream of PKC in the activation of NADPH oxidase in erythrocytes. Data presented are representative of repeated experiments with batched samples. n = 4 each for AA and SS samples in all graphs above, and P < .05 where designated by asterisks.