Core 2 O-glycans on differentially glycosylated CD45 contribute to gal-3 binding. (A) SUDHL-6 (S-6) and SUDLH-9 (S-9) cells express different populations of CD45. Cell lysates were separated on a 3%-8% Tris-acetate SDS-PAGE gel to resolve high-molecular-weight isoforms of CD45, detected by immunoblot. (B) SUDHL-6 and SUDHL-9 cells express equivalent amounts of total CD45 on the cell surface. Cells were analyzed by flow cytometry using the 2B11 + PD7/26 mAbs that recognizes all 5 CD45 isoforms. (C) The abundance of asialo-core 1 O-glycans on the surface of SUDHL-6 and SUDHL-9 cells was determined by binding of biotinylated PNA, detected with avidin-FITC. (D) SUDHL-6 but not SUDHL-9 cells express mRNA encoding the enzyme C2GnT-1, that initiates addition of lactosamine chains to asialo-core 1 O-glycans. C2GnT-1 mRNA detected by RT-PCR. (E) siRNA targeting (+) reduced C2GnT-1 mRNA in SUDHL-6 cells. Nontargeted siRNA (−) was used as a control. Knockdown efficiency was determined 24 hours posttransfection by RT-PCR. (F) Reduction of C2GnT-1 expression by siRNA reduced cell-surface gal-3 association with CD45. Biotin-gal-3 or biotin-BSA was added to SUDHL-6 cells treated with siRNA for C2GnT-1 (+) or control (−). Biotinylated gal-3 and associated binding partners were precipitated from lysates with streptavidin-agarose (SA). While gal-3 precipitated CD45 from control cells, there was a significant reduction in CD45 association with gal-3 in cells with reduced C2GnT-1.