Mouse dimeric sP-selectin-Fc binds with higher avidity to PSGL-1 than mouse monomeric sP-selectin. (A) Schematics of mouse sP-selectin and sP-selectin-Fc. (B) SDS-PAGE of sP-selectin and sP-selectin-Fc under reducing conditions, followed by Coomassie blue staining. (C) Western blots (WB) of sP-selectin and sP-selectin-Fc electrophoresed under reducing and nonreducing conditions, probed with polyclonal anti-P-selectin IgG or mAb HPC4. (D) Elution profiles of the indicated proteins applied to a Superdex 200 column, with fractions detected by absorbance at 280 nm. The column was calibrated by the elution profile of the indicated proteins. (E) Overlays of increasing concentrations of sP-selectin or sP-selectin-Fc binding to 2-GSP-6, a surrogate for PSGL-1, on a sensor surface. The horizontal line indicates when sP-selectin or sP-selectin-Fc in running buffer was injected. (F) Nonlinear curve fits of specific binding data for proteins injected in running buffer (Buffer) or in plasma diluted 1/4 in running buffer (Plasma). The data represent the mean ± standard deviation (SD) from 3 experiments. (G-H) Flow cytometry of mouse neutrophils (G) and monocytes (H) incubated with FITC-labeled sP-selectin or sP-selectin-Fc with or without oligomerization by mAb HPC4, in Ca²⁺-containing buffer or in buffer containing EDTA. The data in panels B-D and G-H are representative of 3 experiments. DF, dye front; mAU, milliabsorbance unit; RU, resonance unit.