Figure 3.

NOX2 assembly and its link to the NOX2 O₂⁻production in dHL60 cells. Formation of the NOX2 complex associated with wt Rac activation and its resultant NOX2 O₂⁻ production in cells was examined. For this analysis, dHL60 cells transfected with wt Rac, constitutively active G12V Rac, or redox inert C18S or C157S Rac were used. A, O₂⁻ production from the PMA-stimulated dHL60 cells was examined by monitoring the oxidation of cytochrome c over time as described in the Experimental procedures section. B, the wt Rac activity change over time in the PMA-stimulated dHL60 cells was examined by using the colorimetric wt Rac activity assay kit (Abcam) as described in the Experimental procedures section. For this analysis, the pull-down NOX2 complexes resuspended in the sample buffer were used. All wt Rac activity assays were performed independently three times; their average values with SD are shown. C, the cellular active NOX2 complex formation in the PMA-stimulated dHL60 cells was examined by using pull-down and Western analyses as described in the Experimental procedures section. For this assay, as noted in the Experimental procedures section, the pull-down NOX2 complexes suspended in the sample buffer were used. Upper panel, the Western analysis of the pull-down sample with gp91 antibody is shown. Middle panel, the Western analyses of the pull-down sample with p40, p47, and p67 antibodies as well as a wt Rac antibody are shown. Lower panel, as loading controls, the cytosolic wt Rac and actin contents in the whole-cell lysates also were analyzed by using wt Rac and actin antibodies. NOX2, NADPH oxidase 2; PMA, phorbol myristate acetate.