Identification of potential L-selectin ligands on KG1a cells. (A) SDS/PAGE (6% reducing gel) and Western blot analysis of HECA-452-reactive membrane proteins (30 μg/lane) from human leukemia cell lines K562 (lane 1), RPMI-8402 (lane 2), HL-60 (lane 3), KG1a (lane 4), neuraminidase-treated KG1a (lane 5), and OSGE-treated KG1a (lane 6). (B) Reexpression of HECA-452-reactive proteins on KG1a whole cells after neuraminidase and then tunicamycin treatment. Lanes: 1, untreated; 2, neuraminidase; 3, neuraminidase then 24-h DMSO recovery; and 4, neuraminidase then 24-h tunicamycin recovery. (C) Autoradiography of KG1a membrane proteins metabolically radiolabeled with 2-[³H]mannose resolved on reducing 6% SDS/PAGE. (D) L-selectin-dependent, lymphocyte tethering and rolling on blotting membrane under hydrodynamic flow conditions (2.3 dynes/cm²).

L-selectin ligand activity of immunoprecipitated CD44. KG1a CD44 immunoprecipitates (Hermes-1 mAb; lanes 3 and 6) resolved on a reducing 9% SDS/PAGE gel, transferred to PVDF membrane, and immunostained with either Hermes-1 or HECA-452. Immunoblots included total cell lysate (lanes 1 and 4) and rat IgG isotype control immunoprecipitate (lanes 2 and 5) groups. L-selectin-dependent lymphocyte rolling was observed in the parallel-plate flow chamber (2.3 dynes/cm²) only over the 98-kDa protein (lymphocyte-binding data presented in parentheses as the mean number of lymphocytes rolling per field at ×200 magnification from more than five different fields).

Effect of chlorate treatment on sulfation and L-selectin ligand activity of KG1a CD44. (A) SDS/PAGE (6%, reduced) autoradiogram of immunoprecipitated CD44 from non-chlorate- and chlorate-treated KG1a cells radiolabeled with [³⁵S]SO₄. (B) HECA-452 immunoblots of immunoprecipitated CD44 from chlorate-treated (+) and non-chlorate-treated (−) KG1a cells. L-selectin ligand activity of the 98-kDa band observed in the parallel-plate flow chamber (2.3 dynes/cm²) was equivalent in sulfated and nonsulfated (chlorate-treated) CD44 (binding data presented in parentheses as the mean number of lymphocytes at ×200 magnification per field; five fields counted).

Western blot analysis of KG1a CD44 with HECA-452 or anti-CD44 mAbs. SDS/PAGE (6% reducing gel) resolution of the 98-kDa gel fragment from the third round of SDS/PAGE purification immunoblotted with either HECA-452 (lane 1) or anti-CD44 mAbs A3D8 (lane 2) and Hermes-1 (lane 3).

Effect of N-glycosidase-F on L-selectin ligand activity of immunoprecipitated CD44 from KG1a cells and from AML (M5) blasts. Immunoprecipitated CD44 from KG1a cells (A) or from AML (M5) blasts (B) were untreated (−) or treated (+) with N-glycosidase-F, resolved on reduced 9% SDS/PAGE, and immunoblotted with HECA-452. L-selectin ligand activity of the 98-kDa band in each case was observed in the parallel-plate flow chamber (2.3 dynes/cm²), and lymphocyte-binding data are presented in parentheses as the mean number of lymphocytes rolling at ×200 magnification; five fields counted.