Figure 7.

Effects of mutations of conserved amino acids on Nox enzymatic activity and formation of the Nox2-p22phox complex. (A) Conserved amino acids (circles) are indicated on a schematic of the Nox domain, and residue numbers corresponding to the human Nox2 protein sequence are keyed in Figure 6. Filled circles indicate known point mutations in individual variants of X-linked CGD. (B) HEK293 cells that constitutively express p22phox were co-transfected with wild type (WT) or the indicated mutations of Nox2 along with p47phox, p67phox, and Rac1(V12G) or with empty vector (mock). Each point mutation of human Nox2 is indicated by the single letter amino acid code. ROS production was measured as described, and the values are presented as mean ± SD (n = 4). These experiments have been repeated three times with similar results. (C) Nox2 and p22phox protein expression was probed by Western blotting (WB) with monoclonal antibodies 54.1 and 44.1, respectively. Proteins were immunoprecipitated (IP) with antibody 54.1 prior to SDS-PAGE. The asterisks indicate IgG heavy chain (* in upper panels) and light chain (** in lower panels). Nox2 protein is expressed as both unglycosylated (65 kDa, immature) and glycosylated (90–100 kDa, mature) forms. p22phox co-immunoprecipitated with Nox2 was seen at 22 kDa. These experiments have been repeated more than three times with similar results.