Differential expression of E-selLs on CD34^pos and CD34^neg subsets isolated from the Lin^neg CD38^neg fraction of human BM. (A) Overview of the gating strategy used to isolate CD34^negCD38^neg and CD34^posCD38^neg fractions by fluorescence-activated cell sorting of lineage-depleted (Lin^neg) BM MNCs. Left panel, dot plot represents the cell surface expression of a lineage marker cocktail (including CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, CD235a, and CD7). Cells residing in the negative fraction (R1) were further gated for CD38-negative cells (R2) (middle panel) and then subdivided into 2 subpopulations based on CD34 expression, CD34^pos and CD34^neg residing in R3 and R4 gates, respectively (right panel). Data shown are representative of n = 4 experiments. (B) Left panel, representative E-Ig staining profile of CD34^pos and CD34^neg subpopulations isolated as depicted in panel A. The shaded curve shows EDTA control (20 mM; on the Lin^negCD38^negCD34^pos subset), whereas dotted red and solid blue curves show the specific binding of CD34^neg and CD34^pos subsets, respectively (n = 4). Middle panel, lysates of CD34^pos BM cells (CD34^pos-BM), Lin^negCD38^negCD34^pos, and Lin^negCD38^negCD34^neg populations isolated from human BM were normalized for total protein level and subjected to western blot analysis. Membranes were blotted with E-Ig, CD44, or β-actin followed by isotype-matched HRP-conjugated mAb for visualization. This is representative of n = 4 independent experiments. Supplemental Figure 1 shows western blots where CD44 was immunoprecipitated from these cell populations and blotted with E-Ig, CD44, and HECA-452. Right panel, flow cytometric analysis of E-selLs expressed on the 2 subpopulations isolated as in panel A is shown as the average percent of expression (above the isotype control) of n = 3 independent experiments. *P < .05 relative to CD34^neg subpopulation. NIH Image J was used to quantify the intensity of western blot bands using the gel analyzer tool; the number displayed represents the density of each band related to the Lin^negCD38^negCD34^neg band as a standard. FSC, forward scatter.