Expression patterns of Plg-Rs on TG-recruited peritoneal macrophages. (A) FACS. Thioglycollate-induced mouse peritoneal cells were incubated with the antibodies to H2B, α-enolase, annexin 2, and p11 (20 μg/mL) for 30 minutes at 4°C and were then stained with FITC-labeled goat anti–rabbit IgG for 30 minutes. Viable macrophages (low scatter, high integrin αMβ2 expression) were analyzed by FACS. Staining with each Plg-R antibody is highlighted with the black line and the light gray area is the reaction with nonimmune rabbit IgG. (B) Cell-surface biotinylation. TG-induced mouse peritoneal macrophages were harvested, subjected to surface biotinylation, captured on streptavidin-agarose, and analyzed by SDS-PAGE, followed by Western blotting with each of the anti–Plg-Rs. (C) Immunofluorescence. Macrophages were reacted for antibodies to each Plg-R or nonimmune rabbit IgG. After washing, the cells were then stained with Alexa 488 antirabbit IgG, fixed in 2% paraformaldehyde, and mounted in mounting medium containing DAPI. Images were captured as in Figure 3. The images shown are representative of numerous areas of the slides. (D) Inhibition of Plg binding to macrophages by Fab to Plg-Rs. Mouse peritoneal macrophages were collected after 72 hours from TG-treated mice. Cells were preincubated with Fab fragments (8 μM) of anti-H2B, anti–α-enolase, anti–annexin 2, anti-p11, or control nonimmune Fab for 30 minutes at 4°C followed by incubation with Alexa 488–labeled Plg (200 nM) at 4°C for 1 hour. The cells were washed and bound Plg was measured by FACS. Specific binding was determined by subtracting the binding values in the presence of EACA. Percentages of specific binding are plotted. Data are means plus or minus SD of 3 independent experiments.