Figure 7
Distribution of exogenously expressed p47phox-PX domain and RhoB during phagocytosis in human neutrophils. Neutrophils were nucleoporated with the expression vectors EGFP-p47-PX or EGFP-RhoB as described in figure 2. After a 2 h recovery period, cells were incubated in the presence of Texas Red-labeled opsonized-zymosan particles at 37°C for 10 or 15 min. Samples were washed, fixed and analyzed by confocal microscopy as described in "Methods." The images were processed and analyzed for the distribution of the fluorescent proteins using the NIH image processing and analysis program IMAGE/J software. The plot profiles, in the right panel, show that EGFP-RhoB is accumulated around the phagosome (red arrows indicating peak of fluorescence intensity) while the plot profiles corresponding to EGFP-p47-PX show a plateau of fluorescence intensity (red lines). The white arrows show the distribution of p47phox-PX domain in membrane protrusions surrounding the opsonized particle at time zero. Scale bar = 3 μm. B, The difference between the fluorescence intensity at the phagosome membrane versus that at the surrounding cytosolic area (ΔFI) was calculated using the NIH image processing and analysis program IMAGE/J software as described under "Methods." The results are mean ± SEM of three phagosomes from different cells.