Effects of p40phox mutations in the PX, SH3, and PB1 domains on translocation of p40phox to the phagosome. (A) Schematic illustration of domains within p40phox and mutants studied for translocation in PLB-985 neutrophils. (B) Time-lapse confocal microscopy was used to monitor translocation of wild-type YFP-p40phox and indicated YFP-p40phox mutants in PLB-985 neutrophils. In the experiments shown, all but YFP-p40phoxR105A were expressed using a transient transfection method. Wild-type YFP-p40phox, YFP-p40phoxR105A, and YFP-p40phoxW207R accumulate on the phagosomal cup but YFP-p40phoxD289A does not. N shows the nucleus and asterisks indicate the IgG-zymosan phagosomes monitored. Bar represents 5 μm. (C) The relative fluorescence intensity compared with the cytosol for 5 phagosomes in PLB-YFPp40 R105A cells at indicated stages is shown in the graphs, as mean plus or minus SD. Internalized indicates 120 seconds after phagosome closure. (D) Phagosomes exhibiting translocation of either YFP-p40phox or YPF-p40phoxR105A in the phagosomal cup were scored for persistence of translocation at 3 minutes after closure. The percentage of phagosomes showing persistent translocation of p40phoxR105A was significantly lower than for YPF-p40phox (*P < .03, n = 11 phagosomes in each group; Fisher exact test). (E-H) A lentiviral vector expressing an shRNA from the 3′ untranslated region of the p40phox cDNA was used to transduce PLB-985, PLB-YFPp40, or PLB-YFPp40R105A cells to generate p40phox knockdown (p40KD PLB-985) cells deficient in endogenous p40phox. (E) Western blot analysis of YFP-tagged proteins and endogenous p40phox, p67phox, and actin in PLB-985 neutrophil lysates. Lane 1 shows p40KD PLB-985; lane 2, p40KD PLB-YFPp40; lane 3, p40KD PLB-YFPp40R105A, and lane 4, PLB-985. A vertical line has been inserted to indicate a repositioned gel lane. (F-H) NADPH oxidase activity in neutrophil-differentiated p40KD PLB-985 cell lines. 1 or ◇ indicates p40KD PLB-985; 2 or ■, p40KD PLB-YFPp40; and 3 or ▵, p40KD PLB-YFPp40R105A. (F) PMA-induced superoxide release detected by isoluminol. The insert represents the mean plus or minus SD (total RLU value over 54 minutes, measured at 1-minute intervals) of 2 independent experiments. (G-H) Extracellular (isoluminol) and intracellular (luminol plus SOD and catalase) superoxide production during synchronized phagocytosis of IgG-Latex beads (G) or IgG-Zym (H). The insert represents the mean plus or minus SD (total RLU value over 54 minutes, measured at 1-minute intervals) of 3 independent experiments.
