Generation of ΔCD mice. (A) Schematic of the extracellular, transmembrane (TM), and cytoplasmic domains of murine PSGL-1. The amino acid sequence of the WT cytoplasmic domain is depicted. *Residues implicated by mutagenesis as contributing to binding to ERM proteins.18 +Residues that directly contact an ERM protein in a crystal structure.41 The 2 arginines in the ΔCD cytoplasmic domain are shown. (B-D) Diagrams of the WT Selplg allele, the targeted allele, and the targeted allele after Cre-mediated deletion of the neo cassette. The probe used for Southern blotting is underlined. (E) Southern blot of BamHI-digested genomic DNA from WT mice and from mice heterozygous or homozygous for the targeted allele. (F) Flow cytometric analysis of PSGL-1 expression on peripheral blood neutrophils from WT mice, ΔCD mice (homozygous for the targeted allele), and PSGL-1 knockout (KO) mice. Cells were incubated with PE-conjugated anti–PSGL-1 mAb 2PH1. (G,H) Western blots of leukocyte lysates electrophoresed under reducing or nonreducing conditions. Blots were probed with rabbit anti–PSGL-1 IgG. Arrows mark the precursor and mature forms of PSGL-1. (I) Recombinant moesin was incubated with GST fused to the entire cytoplasmic domain of PSGL-1 (GST-CD), the cytoplasmic domain in which 4 residues required for binding to ERM proteins were substituted with alanines (GST-CD–mutated), or to the 2 arginines in the cytoplasmic domain of ΔCD mice (GST-RR). Precipitated protein was eluted and probed by Western blot with antimoesin antibody under reducing conditions. Equivalent loading was confirmed by reprobing with anti-GST antibody (data not shown). Results are representative of 3 experiments. (J) Leukocyte lysates or PSGL-1 immunoprecipated from total plasma or from microparticle or soluble fractions of plasma were probed by Western blot with anti–PSGL-1 mAb MTH31 under reducing conditions. Results are representative of 5 experiments.
