FIG. 4.

Analysis of neutrophil membrane-associated NADPH oxidase components after incubation with A. phagocytophila and PMA stimulation. Human neutrophils were incubated in the presence or absence of host-cell-free A. phagocytophila (HGE) for 30 min prior to stimulation with PMA (5 min with 0.5 μg of PMA/ml). Cells were lysed, and membrane fractions were obtained by high-speed centrifugation. Membrane fractions (25 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane prior to being probed with antibodies against gp91^phox (goat), p67^phox, p47^phox, p40^phox, and p22^phox. Western blot results are representative of more than three independent experiments, each performed with neutrophils derived from different donors and freshly prepared host-cell-free A. phagocytophila.