Translocation of p47^phox and p67^phox to plasma membrane in intact cells activated with PMA and in the cell-free oxidase reconstitution assay stimulated with SDS. (A) The indicated cells undergone granulocytic differentiation for 5 days were stimulated with (+) or without (−) PMA (500 ng/mL) for 10 minutes at 37°C, and the plasma membranes were prepared on discontinuous sucrose gradients and analyzed for translocation of p47^phox and p67^phox by immunoblot analysis using p47^phox and p67^phox antibodies (left panel). After stripping, the blots were reprobed with gp91^phox and p22^phox monoclonal antibodies to show an equal loading (right panel). Each lane was loaded with 5 μg of proteins. (B) Membranes separated from the indicated cells were mixed with 3-fold of neutrophil cytosol in the cell-free oxidase reconstitution assay. After 10 minutes of incubation at 25°C in the presence (+) or absence (−) of 100 μmol/L SDS, the membranes were reisolated by ultracentrifugation and detected for the translocation of p47^phox and p67^phox (left panel) by immunoblot analysis as described in (A). Each lane was loaded with 5 μg of proteins.