Western blot analysis of CLA, PSGL-1, and CD43 isolated from human CLA⁺ T cells treated with glycosylation inhibitors.

Cell lysates were prepared from human CLA⁺ T-cell cultures treated with neuraminidase (Neur.) and then grown in the presence of diluent control (PBS), tunicamycin (0.02 mM), swainsonine (0.23 mM), 4-F-GlcNAc (0.05 mM), BAG (2.0 mM), or GlcNAc (5.0 mM, negative control) for 30 hours. Lysates (25 μg per lane) were resolved on 6% reducing SDS-PAGE gels and blotted onto PVDF membrane. Blots were then probed with either antihuman CLA moAb HECA-452 (1 μg/mL; panel A); antihuman PSGL-1 moAbs PL-1, 2G3, and 4D8 (1 μg/mL each; panel B); or antihuman CD43 moAb L60 (1 μg/mL; panel C) and were developed in Western Blue stain. In panel A, note the disappearance of HECA-452–reactive PSGL-1 (CLA) after neuraminidase treatment and after recovery in tunicamycin, 4-F-GlcNAc, and BAG. In panel B, note the marked reduction of PSGL-1 polypeptide in cells recovered in tunicamycin. Also, in panel C, note the reduction inO-glycan–dependent L60 epitopes on CD43 from cells recovered in tunicamycin and BAG with no effect by complexN-glycan inhibitor swainsonine.