Exofucosylation (GPS) of mouse LSK cells enforces HCELL expression, yielding higher E-selectin binding and increased homing to marrow. (A) Flow cytometric analysis of E-Ig binding on BT-LSK and GPS-treated LSK (GPS-LSK) cells. EDTA control displayed a mean fluorescence intensity = 3.1. Results displayed are representative of at least n = 5 flow cytometric experiments. (B) GPS-LSK and BT-LSK cells were lysed, and Western blot analysis was performed on whole cell lysates and blotted with E-selectin and CD44. (C) CD44 was immunoprecipitated (with IM7 and KM114) from equivalent amounts of cell lysates from GPS-treated (+) or untreated (−) mouse LSK cell lysates that had been either treated with N-glycosidase F (+) or not (−). Immunoprecipitates were then electrophoresed and blotted with E-Ig and CD44. Staining with E-Ig was performed in the presence of Ca²⁺. (D) GPS-LSK (black bars) or BT-LSK (white bars) were labeled reciprocally with CFDA-SE and/or CTO and injected intravenously in a 1:1 ratio competitively into C57BL6 mice. Bone marrow was analyzed 16 hours after the injection to determine the percentage of carboxyfluorescein succinimidyl ester and CTO-positive cells present within a defined gate-representing mouse LSK cells. Mice that did not receive cells were used to determine the background signal. Error bars represent the SEM. Data are representative of 6 mice. Parallel control experiments measuring the competition of BT cells to untreated cells did not show any advantage or disadvantage of either cell type (supplemental Figure 4).