HCELL is the predominant E-selectin ligand on human CD34⁺ cells, whereas CLA is the predominant E-selectin ligand on mouse LSK cells. Western blot analysis of CD44, CD43, and PSGL-1 immunoprecipitated from cell lysates of human KG1a cells, human CD34⁺ cells, and mouse LSK cells, stained with E-Ig (A) and HECA-452 mAb (B). As shown here, whole cell lysates of human and mouse cells were initially exhaustively immunoprecipitated using mAb directed to PSGL-1; after clearance of PSGL-1, the residual lysate was exhaustively immunoprecipitated with anti-CD43 mAb and then followed by anti-CD44 mAb. Similar results were obtained when the order of the glycoproteins immunoprecipitated was varied. No detectable signal for PSGL-1, CD43, or CD44 was observed in lysate(s) after removal of the target glycoprotein(s) indicating that immunoprecipitation depleted the respective glycoprotein(s) from lysate(s). The absence of residual HECA-452 and E-Ig reactivity after exhaustive immunoprecipitation indicates that no other glycoprotein(s) appear to be detected as E-selectin ligands within the respective HSPC-enriched populations. Data shown in supplemental Figure 2 corroborate this conclusion. Results shown are representative of 6 separate experiments.