Figure 6.

P47phox and p67phox segregation to low-density detergent (Brij-58) resistant membrane is increased by cell activation, and their membrane translocation is cholesterol-dependent. (A) Brij-58 lysates of control or PMA-stimulated HL60 cells were centrifuged to equilibrium in a sucrose gradient, and then analyzed by Western blotting using antibodies to p67phox, p47phox, or Rac1. (B) Control or cholesterol-extracted HL60 cells were stimulated with PMA, and subsequently the particulate membrane fraction was analyzed for the presence of p67phox, p47phox, or Rac1 by Western blotting. Equal aliquots were also analyzed for p22phox as loading control. (C) HL60 cells were subjected to cholesterol extraction (mβCD), in some cases followed by cholesterol replenishment (mβCD chol), before stimulation with PMA. p67phox, p47phox, Rac1, and p22phox was analyzed by Western blotting as described above. (D) The graph represents mean and s.e. of three independent experiments performed as in (C). Individual p67phox, p47phox, and Rac1 bands were quantitated densitometrically, and the results expressed as percent translocation in cholesterol-extracted (white bars) or cholesterol-replenished (filled bars) cells relative to control cells. (E) HL60 cells stimulated with PMA were subjected to chemical crosslinking with DTSP and the particulate membrane fraction, purified as above, was subjected to sucrose gradient centrifugation. Collected fractions were electrophoresed under reducing conditions and analyzed by Western blotting with anti-p67phox, p47phox, Rac1, gp91phox, and p22phox antibodies.