Analysis of homing-related molecules on murine Flk2⁻CD34⁻ LT-HSCs and Flk2⁻CD34⁺ ST-HSCs illustrates a difference in the expression of E-selectin ligands and CXCR4. (A) Flow cytometric analysis was used to determine sLe^x expression on HSC populations using HECA-452-mAb (gray histogram). Isotype controls (rat IgM) are illustrated in the black histograms. E-selectin binding was also assessed by incubating the cells with E-selectin-hIg chimeric protein followed by anti-human IgG-PE (gray histogram). As a control, 10 mM EDTA was added to abrogate the binding of cells to E-selectin (black histogram). (Ai) Representative histograms are shown from n = 5 independent experiments. A statistical analysis was performed with the unpaired t test (**P = .001 and ***P < .001) as illustrated in (Aii). (B) The expression of the integrins, CD49e, CD49d, and CD29, and the chemokine receptor, CXCR4, on HSC populations was analyzed by flow cytometry. Positive expression was determined as the percent positive cells ± SEM compared with FMO control. Statistical analysis was performed using the unpaired t test (**P = .001). (C) Flow cytometric analysis of E-selectin ligands (CD43, CD44, and PSGL-1) expressed on the 2 subsets of HSCs. Results are expressed as an average percent of expression (above the isotype control) of n = 3 independent experiments. (D) Mass spectrometry data analysis of E-selectin ligands were immunoprecipitated using recombinant E-selectin protein from lysates of Flk2⁻CD34⁺ and Flk2⁻CD34⁻ HSCs, and the purified proteins were identified by mass spectrometry. The identified proteins were analyzed using cell adhesion as a biological process in gene ontology and the data plotted according to the P value for each identified protein.