Binding of fluid-phase murine P-selectin to transfected CHO cells.

(A) Specificity of binding. Left panel: CHO cells expressing Core2GlcNAcT-I, FTVII, and wild-type PSGL-1 were incubated with murine P-selectin/IgM in the presence or absence of EDTA or anti–PSGL-1 mAb 4RA10. Middle panel: The cells were incubated with P-selectin/IgM or murine CD45-IgM. Right panel: The cells were preincubated in the presence or absence of O-sialoglycoprotein endopeptidase (OSGE) and then incubated with P-selectin/IgM. Bound P-selectin/IgM was detected with FITC-conjugated goat antihuman IgM. Control cells were incubated with FITC-conjugated goat ant-human IgM alone. (B) CHO cells expressing Core2GlcNAcT-I, FTVII, and either wild-type or the indicated mutant murine PSGL-1 construct were incubated with murine P-selectin/IgM. Bound P-selectin/IgM was detected with FITC-conjugated goat antihuman IgM. Control cells were incubated with FITC-conjugated goat antihuman IgM alone. The data are representative of at least 5 experiments.