NADPH oxidase activity in neutrophil-differentiated PLB-985 cells expressing YFP-phox proteins during phagocytosis of IgG-zymosan. (A) Western blot analysis of exogenous YFP-tagged phox proteins and endogenous phox proteins in PLB-985 neutrophil lysates. Lane 1 shows PLB-p67YFP; lane 2, PLB-985; lane 3, PLB-YFPp40; and lane 4, PLB-985. (B-E) NADPH oxidase activity in PLB-p67YFP neutrophils during synchronized phagocytosis of IgG-zymosan was quantified using isoluminol in the presence of HRP (B,C) to detect extracellular superoxide or luminol in the presence of SOD and catalase (D,E) to detect intracellular activity. Assays were performed in the absence or presence of preincubation with 100 nM wortmannin, as indicated. Representative kinetic plots (B,D) and mean plus or minus SD of relative integrated RLU data are shown (3 assays). The activity profile was similar for PLB-p67YFP and PLB-YFPp40 cells (data not shown). ♦ indicates no wortmannin; ◇, 100 nM wortmannin.