Colocalization of Plg-R with Plg on the surface of J774.1 cells. Localization of Plg with either H2B, α-enolase, annexin 2, or p11 on the cell surface of J774A.1 cells assessed by confocal microscopy. The cells were grown on coverslips, serum starved, and incubated with Plg (200 nM). After washing, the cells were incubated with a combination of rabbit anti–Plg-R (20 μg/mL) and rat monoclonal anti-Plg (20 μg/mL) for 30 minutes at 4°C in 1.5% BSA-HBSS (A-L). Nonimmune rabbit IgG and rat IgG served as controls. Cells were washed and then stained with Alexa 488 antirabbit and Alexa 568 antirat antibodies. Cells were fixed in 2% paraformaldehyde and mounted in Vectashield DAPl (4′,6-diamidine-2′-phenylindole dihydrochloride) mounting medium (Vector Laboratories, Burlingame, CA). Images were captured by confocal microscopy (Leica TCS-SP2 laser scanning confocal microscope) with 40×/1.25 NA oil-objective lens at RT and photographed using Leica confocal software v.2.61 (Leica Microsystems, Heidelburg, Germany). All images were processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). In separate experiments (M-P), J774A.1 cells were treated with CpB (10 U/mL), washed, and then incubated with Plg (200 nM). Cells were further washed and stained for bound Plg and Plg-R as described for panels A-L. Colocalization of Plg (red) with the cell-surface Plg-Rs (green) is detected as yellow in the merged images (C,F,I,L). Upon CpB treatment, the predominant staining seen in merged images (M-P) was only green, showing that Plg bound to cell-surface H2B, α-enolase, annexin 2, or p11 via their C-terminal lysines. Insets of panels M-P show the anti-Plg staining upon CpB treatment. The images shown are representative of numerous areas on the slides.