Fig. 4. Effect of the D289A substitution in the PC motif of p40^phox on its interaction with p67^phox both in vivo and in vitro. (A) Direct interaction of p40^phox with p67^phox. GST-tagged PB1 domain of p67^phox (GST–p67-PB1) or GST alone was incubated with MBP–p40^phox (WT) or MBP–p40^phox (D289A) and proteins were pulled down with glutathione–Sepharose-4B. The precipitated proteins were subjected to SDS–PAGE, followed by staining with CBB, as described, see Materials and methods. Positions for marker proteins are indicated in kDa. (B) Expression of p40^phox (D289A) in K562 cells. K562 cells stably expressing gp91^phox, p67^phox and p47^phox were transfected with pREP4 encoding p40^phox with the D289A substitution, p40^phox (D289A). The K562 cells were stained with anti-p40^phox serum (filled histogram) or pre-immune serum (open histogram) and analyzed by flow cytometry. (C) Interaction of p40^phox with p67^phox in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with an anti-p67^phox or control IgG (cont.) (left panel), or an anti-p47^phox antibody or control IgG (cont.) (right panel) followed by immunoblot with the indicated antibody, as described under Materials and methods.