E-selectin binds predominantly to O-glycans on CD34. (A) 4H11-mAb (4964 RU) or its isotype control (4036 RU) were immobilized to capture CD34 from KG1a lysates that were treated with neuraminidase (Treated; blue line) or left untreated but subjected to the same buffer-treatment conditions, incubation times, and temperatures (Control; red line). Following CD34 capture, E-Ig was injected at 354 nM. The same surface was used for both the treated and the control binding studies with a surface regeneration step between the 2 runs. The normalized (red dashed line) sensorgram is the same as the control but normalized to the treated sensorgram based on the ratio of accumulated CD34 RU prior to E-Ig injection. (B) For western blot analysis, equivalent amounts of CD34 immunoprecipitates from HSPC-enriched lysates (KG1a or CD34^pos-UCB) were either treated with neuraminidase (+) or not (−) and blotted with either E-Ig (top panel) or CD34 (QBend-10, lower panel). Note that the apparent increase in MW of CD34 is attributed to the loss of the negatively charged sialic acid. (C) SPR analysis of the PNGase F treatment was performed as in panel A using 4H11-mAb (6500 RU) or its isotype control (5810 RU). k_off and k_off-apparent were calculated as described in Figure 3B. (D) CD34 immunoprecipitates were treated with PNGase F and subjected to western blotting for E-Ig (top panel) or CD34 (lower panel) as in panel B. (E) SPR analyses of OSGE treatment were performed as in panels A and C using 4H11-mAb (9000 RU) or its isotype control (6500 RU). (F) Western blot analysis of the treated CD34 immunoprecipitates were performed as in panels B and D. CD34 (Qbend-10) was used as an internal control to confirm N- and O-glycan removal. The sensorgrams presented are corrected for the bulk refractive index and nonspecific interactions using the isotype controls. All results are representative of n = 3 independent experiments.