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. 2021 Jul 20;297(2):100982. doi: 10.1016/j.jbc.2021.100982

Figure 6.

Figure 6

Effect of Vav and O2−on the initiation of the NOX2 autoactivation. The effect of Vav and exogenous O2− on the initiation of activation of the hysteretic wt Rac and NOX2 was examined by using the Vav knocked-down dHL60 cells. Preparation and quantification of exogenous O2− were described in the Experimental procedures section. A, O2− production from the PMA-stimulated Vav knocked-down dHL60 cells in the presence and absence of exogenous O2− (∼2 μM) was examined by monitoring the oxidation of cytochrome c over time as described in the Experimental procedures section. As a control, dHL60 cells that express Vav also were used. B, the wt Rac activity change in the PMA-stimulated Vav knocked-down dHL60 cells in the presence and absence of exogenous O2− (∼2 μM) was as described in B of Figure 3. As a control, dHL60 cells that express Vav also were used. Three independent assays were performed, and the average values with SD are shown. C, upper panel, the wt Rac binding to the NOX2 phox complex in Vav knocked-down dHL60 cells over time in the presence and absence of exogenous O2− was examined by using pull-down and Western analyses. Middle panel, lack of the Vav expression in the Vav knocked-down dHL60 cells also was examined by using Western analysis. For this analysis, the whole-cell lysate of Vav knocked-down dHL60 cells was used. As a control, a Vav Western analysis for dHL60 cells that express Vav also was used. Lower panel, total wt Rac and actin content in the whole-cell lysates of Vav knocked-down dHL60 cells are shown as loading controls. NOX2, NADPH oxidase 2; PMA, phorbol myristate acetate.