Flow-based binding assays confirm that CD34 is a functional E-selL. (A) Raw data depicting the capturing of E-selectin ligand from KG1a lysate on surface-immobilized antibodies and their subsequent binding to E-selectin by SPR. The lanes of the flow cell represent blank (red line), immobilized CD34-mAb (4H11) (7360 RU; red dashed line), immobilized CD44-mAb (Hermes3) (5200 RU; blue dashed line), and immobilized isotype control (4377 RU; blue line). The mAb immobilization (step 1) is not shown. Lysate injection, arrows mark the start and end of the lysate injection, which is then followed by a buffer washing step. The sensorgrams represent the raw data of the uncorrected RU for the buffer bulk refractive index and nonspecific interactions. Inset, western blot analysis and staining for either CD34 (left panel) or PSGL-1 (right panel) after eluting them from the chip confirming specificity of their capturing by SPR. E-Ig/EDTA injection, the injection involves E-Ig (177 nM) in the presence of EDTA (5 mM) as a control to show specificity of E-Ig to its ligands. E-Ig/Ca²⁺ injection, the injection involves Ca²⁺ (1 mM) to support E-Ig (177 nM) binding to its ligands. (B) Corrected sensorgrams for the buffer refractive index and nonspecific interactions. Data were presented as described in panel A but after subtracting the RU of the buffer refractive index and nonspecific interactions from the standard reference flow cell that contains the isotype control. Furthermore, normalization was applied to even out the difference in the level of mAb-captured CD34 and CD44 as described in the supplemental Materials and methods. k_off is the dissociation rate constant for CD34 and CD44 from their respective mAb, and k_off-apparent is the apparent dissociation rate constant for E-Ig, CD34/E-Ig, or CD44/E-Ig from their respective mAb as well as E-Ig from the complexes CD44·Hermes-3-mAb or CD34·4H11-mAb (n = 3 independent experiments). (C) Immunoprecipitations of CD34 were prepared from lysates of CD34^pos-BM and KG1a cells and spotted on glass slides to test for CHO-E binding using a Stamper-Woodruff assay. Adherent CHO-E cells were counted by light microscopy using an ocular grid under magnification ×20. The data are representative of 1 experiment, and the error bars indicate the standard error of the mean (SEM) of 7 fields per slide on 2 slides for each experiment (n = 3 independent experiments). (D) Blot-rolling assays were performed on western blots of CD34, CD44, CD43, and PSGL-1 immunoprecipitates from KG1a cells stained for HECA-452. CHO-E cells were subsequently perfused over immunoprecipitated glycoproteins at 0.25 dyne/cm². After cell perfusion, the numbers of rolling cells per field were counted (red bars). As a control, CHO-E cells incubated with EDTA (blue bars) or mock-transfected CHO cells (CHO-M) (light green bars) were used. Results shown reflect the average number of rolling cells over the HECA-452 blots of n = 7 membrane preparations from 4 distinct fields of view each. Data are mean ± SEM (error bars). *P < .05; **P < .01; ***P < .001.