Cell-surface expression of H2B, α-enolase, annexin 2, and p11 on RAW 264.7 and J774A.1 cells. (A) Cell-surface biotinylation. RAW 264.7 and J774A.1 (i-iv) were dissociated from culture flasks, and cell-surface proteins were labeled with sulfo-NHS-biotin at 4°C for 30 minutes. Cells were lysed and equal amounts of surface-biotinylated proteins were precipitated with streptavidin-agarose, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 12.5% gels) under reducing conditions, and immunoblotted with anti-H2B (i), anti–α-enolase (ii), anti–annexin 2 (iii), or anti-p11 (iv). Each antibody recognized a single major protein with a mobility appropriate for the target antigen. (B) FACS analysis. RAW 264.7 (top panels) and J774A.1 (bottom panels) cells were stained with antibodies to the indicated Plg-R (20 μg/mL) for 30 minutes at 4°C in HBSS-BSA buffer. Cells were then stained with FITC-conjugated goat anti–rabbit IgG. Staining with this antibody is indicated with the black line, and nonimmune IgG is shown by light gray areas. Mean fluorescence intensity (MFI) values, after subtracting the MFI values of the control antibody, are displayed in each histogram. Results are representative of 3 independent experiments and show that each Plg-R is present on the surfaces of these cells.