The sialomucin CD34 is a novel ligand for E-selectin. (A) CD34 was immunoprecipitated from HSPC-enriched lysates of CD34^pos cells isolated from normal UCB cells (CD34^pos-UCB), normal BM cells (CD34^pos-BM), AML BM cells (CD34^pos-AML), or KG1a cells (n = 3 patient samples and KG1a cells). Lysates were subjected to western blot analysis with E-Ig chimera. This is a representative blot of n = 3 experiments. (B) The reciprocal immunoprecipitation assay was performed where E-Ig chimera was used first for immunoprecipitation prior to western blot analysis using CD34 QBend-10 mAb (n = 3). (C-D) AML cells express a unique form of CD34 that does not function as an E-selL. Multiple rounds of E-Ig immunoprecipitations were performed on both normal and AML sample cell lysates, and following the clearance of E-Ig reactive bands, the residual lysates were immunoprecipitated using QBend-10 (CD34-mAb that recognizes all classes of CD34) and subjected to western blot analysis for CD34 (QBend-10) (C) and anti-sLe^x (HECA-452) (D). SN = supernatant. For panel D, as described in the supplemental Materials and methods, only the first elution after E-Ig immunoprecipitation and the CD34 immunoprecipitation are shown. These blots are representative of n = 4 separate experiments. (E) KG1a cells were pretreated with E-Ig chimera or left untreated prior to lipid raft staining with choleratoxin-β (CTB)-AF-594 (green). Fixed cells were then stained with CD34 (Cy5; red) and AlexaFluor-488 streptavidin (blue) toward biotinylated anti-human-Ig to detect E-Ig. The colocalized mask was analyzed using Imaris Coloc software. Cell surface labeling with an isotype control or a secondary antibody alone served as background controls (data not shown). Results are representative of n = 3 independent experiments, 7 fields per experiments; >5 cells per field. Scale bar is 5 μm. (F) CD34^pos cells from AML BM (lower panels) express a CD34 glycoform that does not bind E-selectin, whereas CD34^pos from normal BM (upper panels) counterparts does not. CD34^posCD38^neg lineage-depleted BM cells from either AML or normal BM were prepared for confocal analysis and stained for E-selectin chimera (E-Ig; red), sLe^x expression (HECA-452; green), and CD34 (8G12; blue). Bright field images are also shown. Colocalization analysis was performed using Imaris 7 software to construct the colocalization mask (white). Yellow arrows in the AML images (lower panels) point to spots where CD34 expression is not overlaid with E-selectin binding or sLe^x expression. Results are representative of n = 3 independent experiments. Scale bar is 10 μm. For clarity, panels A-D were performed on CD34^pos samples from normal or AML BM wherever stated and panel F was performed on Lin^negCD38^negCD34^pos cells from normal or AML BM. IP, immunoprecipitation.