FIG. 5.

Effect of GT on in vitro assembly of flavocytochrome b₅₅₈ with cytosolic components. (A) Analysis of p67^phox and p47^phox association with flavocytochrome b₅₅₈. HTG-solubilized membranes (2.5 pmol of heme equivalent) were incubated with rp67^phox (3 pmol) and rp47^phox (8.5 pmol) in the presence (+) or absence (−) of 30 nmol of GT for 5 min and subsequently stimulated with 27.5 nmol of MA for 10 min at room temperature. The mixture was then subjected to immunoprecipitation (IP) with anti-p47^phox serum or normal serum and analyzed by immunoblotting plus development with o-dianisidine (see Materials and Methods). (B) Analysis of Rac2 membrane translocation. Membranes (6 pmol of heme equivalent) were combined with rp67^phox (36 pmol), rp47^phox (252 pmol), and GTPγS-loaded Rac2 (252 pmol) in 0.4 ml of activation buffer in the presence or absence of 60 nmol of GT for 5 min at room temperature. The mixture was then stimulated with 80 nmol of SDS for an additional 5 min, followed by loading onto a discontinuous sucrose gradient (15% over 50%) and centrifugation (see Materials and Methods). The membrane pellet containing assembled NADPH oxidase was recovered and analyzed by immunoblotting with anti-Rac2 serum, followed by detection with an ECL-Plus reaction. Lane 5 shows the addition of GT 5 min after the SDS stimulation, and lane 6 was loaded with 2 pmol of recombinant Rac2. The data are representative of two (A) or three (B) experiments.