Figure 3.

Affinity precipitation of cytochrome b₅₅₈ by GST-p67^PHOX. Eighty picomoles of GST or full-length GST-p67^PHOX were incubated in the presence of 4.3 pmol of cytochrome b₅₅₈ in 200 μl of 20 mM Hepes, pH 7.5/1% Nonidet P-40/10 mM NaCl/1 mM EGTA for 2 h at room temperature. Forty microliters of glutathione-Sepharose 4B beads (Amersham Pharmacia) then were added, followed by incubation for 2 h at 4°C. After washing five times with the same buffer, the beads were pelleted, resuspended in 40 μl of Laemmli sample buffer (22), and boiled for 5 min. Proteins were resolved by 12% SDS/PAGE and transferred onto nitrocellulose. The proteins then were detected by using a monoclonal anti-gp91^PHOX antibody.