FIGURE 1.

H₂O₂ induces EE targeting of p40^phox. A, transfected GFP-p40^phox is localized in the cytoplasm (left); in contrast, GFP-p40^phox(PX) (center; arrow) and p40^phox(F320A) (right; arrow) are localized at dotlike structures, in resting WT HEK293 cells. Bar, 10 μm. B, p40^phox(F320A) co-expressed with p47^phox + p67^phox supports moderate constitutive (32.0 ± 6.2%) and high BIgG-stimulated ROS production in HEK293^{Nox2/FcγRIIa} cells when compared with cells expressing WT p40^phox + p47^phox + p67^phox. Western blotting detects comparable levels of cytoplasmic Phox proteins in both transfection experiments. C, in response to exogenous 1 mm H₂O₂ (120 s after stimulation), GFP-p40^phox expressed in WT HEK293 cells translocates to dotlike structures (arrowheads). D, the dotlike structures are co-stained by an EE marker, EEA1 (arrowheads). E, cytoplasmic GFP-p67^phox co-expressed with mKO-p40^phox in WT HEK293 cells translocates to dotlike structures (arrowheads) after stimulation (110 s) with 1 mm H₂O₂. Similar effects of H₂O₂ are observed in RAW 264.7 cells (supplemental Fig. 3). F, effect of H₂O₂ on the PX-PB1 domain interaction in vitro. The interaction between purified His₆-p40^phox(PX) and GST-p40^phox(PB1) proteins detected by pull-down assays is weakened by the addition of 0.01 mm H₂O₂. Similar results are obtained in three independent experiments. G, effect of H₂O₂ on binding of p40^phox to PI(3)P in vitro. The interaction between purified full-length His₆-p40^phox protein and PI(3)P-containing liposomes detected by pull-down assays is strengthened by the addition of 0.01 mm H₂O₂. Similar results were obtained in three independent experiments. Error bars, S.E.