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Fig. 3. Immunodetection of the protein components of the O2–-generating NADPH oxidase in membrane and cytosolic fractions from murine DCs and bovine neutrophils. 5x106 cell equivalents of DC and neutrophil membrane and cytosol fractions were subjected to SDS-PAGE and then to western blot, using antibodies directed to the two subunits, Nox2 and p22phox, of the membrane-bound flavocytochrome b558 and to the cytosolic factors of oxidase activation, p67phox, p47phox and p40phox. (A) Immunoblots of murine DC membranes, sDC membranes and bovine neutrophil (N) membranes revealed with anti-Nox2 and anti-p22phox antibodies. In this experiment, sDC was obtained from spleens of mice pretreated with Flt-3L. Molecular mass markers (kDa) are indicated. (B) Immunoblots of murine DC cytosol revealed with anti-p67phox, anti-p47phox and anti-p40phox antibodies. (C) Immunoblots of DC membranes showing that p67phox, p47phox and p40phox are detected in crude DC membranes, but not in DC membranes purified on sucrose gradient.





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