Figure 4
Figure 4. Assembly and activation of the NADPH oxidase in response to IgG-SRBCs is fully dependent on binding of p40phox to PtdIns3P and on Rac2. (A) BMNs from WT, p40phoxR58A/R58A, or Rac2KO genetic backgrounds were preincubated with NBT before incubation with IgG-SRBCs and processed as described in Figure 1, with the exception of Rac2KO samples, which were cytospun onto coverslips. Shown are representative images, with IgG-SRBC phagosomes indicated by stars. (B) Primed BMNs from WT (black squares/bar) or p40phoxR58A/R58A genotypes (dark gray diamonds/bar; i,iii); or from WT (black squares/bar), Rac1KD (gray diamonds/bars), Rac2KO (light gray triangles/bars), and Rac1KD/Rac2KO (open circles/bars) genotypes (ii-iii), were subjected to luminol-dependent chemiluminescence assays for total ROS generation as described in Figure 3A. Shown are data (mean ± range) from 1 experiment representative of at least 3, expressed as RLU/s (i-ii), as well as accumulated light emission (ROS response) over 20 minutes for a combination of ≥ 3 experiments (mean ± SEM), expressed as a percentage of the response in WT mouse neutrophils (iii). *P < .0001, paired Student t test (WT/R58A), 1-way ANOVA (WT, Rac1KD, Rac2KO, Rac1KD/Rac2KO) compared with WT response. (C) For quantification of cytosolic fluorescence (1), fluorescence at sites of attachment of IgG-SRBCs to neutrophil (2), around the phagocytic cup (3), the phagosome (4), and after phagocytosis (5), widefield epifluorescence microscopy was used. Mouse BMNs expressing p67phox WT-GFP on a WT or a p40phoxR58A/R58A genetic background or expressing p67phox H69E-GFP on a WT genetic background were primed and incubated with IgG-SRBCs as described in Figure 2C. Images were recorded and presented as described in Figure 3C. Quantification was performed using ImageJ software, as described in “Live imaging” and supplemental Figure 9. Data are presented as a ratio of MFI of phagosome:cytosol. The gray transparent area represents a ratio of phagosome:cytosol of 1.0 or lower, representing no phagosomal translocation. Data are mean ± SEM (n = 4-17 neutrophils). *P ≤ .01 as determined by Student t test between WT and R58A, and between WT and H69E.

Assembly and activation of the NADPH oxidase in response to IgG-SRBCs is fully dependent on binding of p40phox to PtdIns3P and on Rac2. (A) BMNs from WT, p40phoxR58A/R58A, or Rac2KO genetic backgrounds were preincubated with NBT before incubation with IgG-SRBCs and processed as described in Figure 1, with the exception of Rac2KO samples, which were cytospun onto coverslips. Shown are representative images, with IgG-SRBC phagosomes indicated by stars. (B) Primed BMNs from WT (black squares/bar) or p40phoxR58A/R58A genotypes (dark gray diamonds/bar; i,iii); or from WT (black squares/bar), Rac1KD (gray diamonds/bars), Rac2KO (light gray triangles/bars), and Rac1KD/Rac2KO (open circles/bars) genotypes (ii-iii), were subjected to luminol-dependent chemiluminescence assays for total ROS generation as described in Figure 3A. Shown are data (mean ± range) from 1 experiment representative of at least 3, expressed as RLU/s (i-ii), as well as accumulated light emission (ROS response) over 20 minutes for a combination of ≥ 3 experiments (mean ± SEM), expressed as a percentage of the response in WT mouse neutrophils (iii). *P < .0001, paired Student t test (WT/R58A), 1-way ANOVA (WT, Rac1KD, Rac2KO, Rac1KD/Rac2KO) compared with WT response. (C) For quantification of cytosolic fluorescence (1), fluorescence at sites of attachment of IgG-SRBCs to neutrophil (2), around the phagocytic cup (3), the phagosome (4), and after phagocytosis (5), widefield epifluorescence microscopy was used. Mouse BMNs expressing p67phox WT-GFP on a WT or a p40phoxR58A/R58A genetic background or expressing p67phox H69E-GFP on a WT genetic background were primed and incubated with IgG-SRBCs as described in Figure 2C. Images were recorded and presented as described in Figure 3C. Quantification was performed using ImageJ software, as described in “Live imaging” and supplemental Figure 9. Data are presented as a ratio of MFI of phagosome:cytosol. The gray transparent area represents a ratio of phagosome:cytosol of 1.0 or lower, representing no phagosomal translocation. Data are mean ± SEM (n = 4-17 neutrophils). *P ≤ .01 as determined by Student t test between WT and R58A, and between WT and H69E.

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