Direct binding of SSL5 to PSGL-1. (A) PSGL-1–transfected (•) and control (♦) CHO cells were incubated with 0.3 to 10 μg/mL HIS-SSL5 for 30 minutes on ice. Bound HIS-SSL5 was detected with FITC-conjugated anti-Xpress. The data represent mean fluorescence of detected HIS-SSL5 and are mean values ± SEMs of 3 independent experiments. (B) Biotinylated F(ab′)₂ goat anti–human Fcγ antibodies were immobilized (2.5 kRU) onto 2 adjacent channels of a streptavidin-coated sensor chip. Purified recombinant rPSGL/Ig (0.3 mg/mL) was then applied to the second of these channels to reach a density of 0.25 kRU, whereas channel 1 was used as a reference. The response of SSL5 at equilibrium was determined and plotted against the concentration applied. (C-D) CHO–PSGL-1 cells were cultured in the absence or presence of 50 mM sodium chlorate (NaClO₃). Subsequently, cells were tested for binding of HIS-SSL5 (C) and P-selectin/Fc (D). The data represent mean fluorescence of detected HIS-SSL5 and P-selectin/Fc and are mean values ± SEMs of 3 independent experiments. (E) CHO–PSGL-1 cells were treated without (▪) or with (□) 0.2 U/mL neuraminidase for 45 minutes at 37°C. After washing, binding of HIS-SSL5, P-selectin/Fc, KPL1, and anti-CD15s was examined. The data represent relative fluorescence of detected protein and are mean values ± SEMs of 3 independent experiments.