Figure 1.

Localization of gp91^phox and p22^phox in WT and transgenic COS7 cells. (A) Cells were labeled with mAb 7D5, which recognizes an extracellular epitope of gp91^phox, or mAb 449, which recognizes an intracellular epitope on p22^phox. For staining with mAb 449, the cells were permeabilized with saponin as described under Materials and Methods. After incubation with a fluorescein isothiocyanate-conjugated secondary antibody, cells were observed by confocal microscopy. Mouse IgG1 was used in parallel as an isotype control in the cell staining. (Imaging amplifications: ×360 for 7D5 and 449 antibody staining and ×148 for IgG1 staining.) (B) Cellular membrane (Left) and cytosolic fractions (Right) prepared from the indicated COS7 cells were analyzed for gp91^phox and p22^phox expression by immunoblotting with a mixture of mAbs for gp91^phox (24) and p22^phox (24). Each lane was loaded with 10 μg of protein. The band ≈44 kDa in the COS7 p22 and COS7 gp91/p22 lanes represents dimeric aggregate of p22^phox.