Autoradiography of PSGL-1 isolated from metabolically radiolabeled CLA+T cells treated with glycosylation inhibitors.
Radiolabeled lysates were prepared from human CLA+ T-cell cultures treated with neuraminidase (Neur.) and then regrown in [35S]-protein labeling mix (100 μCi/mL [3.7 MBq/mL]) in the presence of PBS (diluent control), 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. PSGL-1 was then immunoprecipitated from lysates (200 μg) with mouse IgG isotype control or antihuman PSGL-1 moAbs PL-1, 2G3, 4F9, and 4D8 (2 μg/mL each) from lysates (200 μg) and resolved on a 6% reducing SDS-PAGE gel. Autoradiography reveals that tunicamycin markedly inhibited PSGL-1 expression, while BAG treatment slightly reduced PSGL-1 expression and mobility of the 140-kDa monomer form. Also, 4-F-GlcNAc did not affect PSGL-1 expression, while it did reduce the molecular weight of the monomer (to 130 kDa).
