Plg-RKT is dispersed over the cell surface and colocalizes with uPAR. M-CSF–differentiated Hoxa9-ER4 cells were grown on coverslips and incubated with a combination of polyclonal rabbit anti–Plg-RKT IgG (20 μg/mL) and mouse monoclonal anti-uPAR (20 μg/mL; A). Cells were washed, fixed in 1% formaldehyde, and then stained with a combination of Alexa 488-F(ab′)2 fragment of goat anti–rabbit IgG and Alexa 568-F(ab′)2 fragment of goat anti–mouse IgG for 60 minutes at 20°C in PBS containing 0.001% Triton X-100. Controls are samples incubated without first antibody. In panel B, cells were preincubated with either PBS (− plasminogen) or 2μM plasminogen (+ plasminogen) for 10 minutes at 4°C. Then, the cells were fixed in 1% formaldehyde, washed, and then stained with polyclonal anti-Plg IgG or mouse anti–Plg-RKT mAb and stained with a combination of Alexa 488-F(ab′)2 fragment of goat anti–rabbit IgG and Alexa 568-F(ab′)2 fragment of goat anti–mouse IgG. Cells were washed and mounted in Immuno-Fluore Mounting Medium. Images were captured using a Zeiss laser confocal scanning microscope, then imported into LSM Examiner and ImageJ for further processing as described in “Scanning confocal microscopy.” The data in panel C were quantified and the number and size of each labeled aggregate were determined as described in “Scanning confocal microscopy.” The results reflect counts (C) and colocalization correlation coefficient (M1) values (last column in panels A-B) from more than 40 cells in 2 independent experiments. Data represent mean ± SEM. *P < .001.
