Effect of shRNA knockdown of p40^phox on NADPH oxidase activity in neutrophil-differentiated PLB-p67YFP cells. PLB-p67YFP cell lines induced for neutrophil differentiation were analyzed by Western blot (A) for the expression of NADPH oxidase subunits and for NADPH oxidase activity during phagocytosis of IgG-zymosan (B-E). Lane 1 shows PLB-p67YFP-pSuper(neo) (empty vector) cells; lane 2, PLB-p67YFP p40^phox knockdown cells. NADPH oxidase assays were performed using isoluminol in the presence of HRP (B,C) to detect extracellular superoxide or luminol in the presence of SOD (D,E) to detect intracellular activity. Representative kinetic plots (B,D) and mean plus or minus SD of relative integrated RLU data are shown (2 isoluminol assays and 4-6 luminol assays). ♦ indicates PLB-p67YFP; ◇, PLB-p67YFP with p40^phox knockdown. (F) Time-lapse confocal images of p67^phox-YFP in p40^phox knockdown cells (Video S5). The relative fluorescence intensity compared with the cytosol for 5 phagosomes at indicated stages is shown in the graph, as mean plus or minus SE. N shows the location of nucleus and asterisks indicate the IgG-zymosan phagosomes monitored. Bar represents 5 μm.