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Blood, Vol. 112, Issue 9, 3867-3877, November 1, 2008
Fc R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome
Blood Tian et al.
112: 3867
Supplemental materials for: Tian et al
Files in this Data Supplement:
- Figure S1. PMA-stimulated superoxide production in COS-7-derived cell lines (JPG, 44.6 KB)
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Integrated RLU values for luminol assays of indicated cell lines (mean ± SD, n = 3) (total RLU value over 42 min, measured at 1 min intervals).
- Figure S2. Distribution of p67phox-YFP and p47phox during IgG-SRBC-phagocytosis in COS-WF, COS-PF-p67YFP, and COSPF40-p67YFP cells as described in Fig. 2 (JPG, 65.9 KB)
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IgG-SRBC was pre-labeled with goat Alexa-633 anti-Rabbit IgG (Invitrogen Molecular Probes). Simultaneous imaging of p67phox-YFP and p47phox following immunofluorescent staining with anti-p47phox and Alexa555-goat-anti-mouse IgG. "Merge" image is the merge of the bright-field and all the fluorescent channels for images of COS-WF. N shows the location of nucleus and asterisks indicate the IgG-SRBC phagosomes.
- Figure S3 (JPG, 65.6 KB)
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(A) PMA- (♦) and IgG-Zym- (◇) stimulated NADPH oxidase activity in the presence of isoluminol and HRP as described in Figure 3B. (B) PMA-induced NADPH oxidase activity assays in the presence of isoluminol and HRP were performed in the absence or presence of pre-incubation with 100 nM wortmannin, as indicated. ♦, no wortmannin; ◇, 100 nM wortmannin. Insert shows the mean ± SD of relative integrated RLU data are shown (3 assays).
- Figure S4. Western blot analysis of exogenous YFP-tagged p40phox proteins and endogenous p40phox, p67phox, and p47phox in neutrophil - differentiated PLB-985 cells (JPG, 71 KB)
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Undifferentiated PLB-985 cells were transduced with retroviral vectors MSCV-YFPp40phox, MSCV-YFP-p40phoxR105A, MSCV-YFP-p40phoxW207R or MSCV-YFP-p40phoxD289A and sorted by flow cytometry for fluorescence. Samples shown were prepared after neutrophil differentiation, and represent the Triton-X-100-soluble cell lysate and the Triton-X-100-insoluble cell pellet.
- Video 1. Time-lapse movie of PLB-p67YFP cells corresponding to the sequence shown in Figure 4A (MOV, 537 KB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~4.5 min.
- Video 2. Time-lapse movie of PLB-YFPp40 cells corresponding to the sequence shown in Figure 4B (MOV, 598 KB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~4.5 min.
- Video 3. Time-lapse movie of wortmannin-treated PLB-p67YFP cells corresponding to the sequence shown in Figure 4C (MOV, 239 KB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~3.75 min.
- Video 4. Time-lapse movie of wortmannin-treated PLB-YFPp40 cells corresponding to the sequence shown in Figure 4C (MOV, 1.10 MB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~7 min.
- Video 5. Time-lapse movie of PLB-p67YFP-p40siRNA cells corresponding to the sequence shown in Figure 5F (MOV, 172 KB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~3.75 min.
- Video 6. Time-lapse movie of PLB-YFPp40/Cherry-PX cells corresponding to the sequence shown in Figure 6A and showing YFPp40 (MOV, 605 KB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~5 min.
- Video 7. Time-lapse movie of PLB-YFPp40/Cherry-PX cells corresponding to the sequence shown in Figure 6A and showing Cherry-PX (MOV, 1.02 MB)
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Sequences were exported as Quicktime movies or sequential TIF images from Metamorph, and final movies were rendered in Adobe Premier. Duration of the clip is ~5 min.
- Video 8. Time-lapse movie of PLB-985 p40phox knockdown cells stably expressing YFP-p40phoxR105A (MOV, 366 KB)
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Sequences were exported as Quicktime movie or sequential TIF images from Metamorph, and final movie were rendered in Adobe Premier. Duration of the clip is ~150 sec.
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