Autoradiography of PSGL-1 immunoprecipitated from human CLA⁺ T cells metabolically radiolabeled with 4-F-Glc[³H]NAc.

Radiolabeled lysate was prepared from human CLA⁺ T-cell cultures grown in 4-F-Glc[³H]NAc (0.2 mM or 3.4 μCi/mL [0.1258 MBq/mL]; 16 μCi/μmol [0.592 MBq/μmol]) for 36 hours. PSGL-1 was then immunoprecipitated from radiolabeled lysate (2 mg) with mouse IgG isotype control or antihuman PSGL-1 moAbs (PL-1, 2G3, 4F9, and 4D8, 2 μg for each 100 μg lysate), resolved on a 6% reducing SDS-PAGE gel, and exposed to film for 28 days. Autoradiography revealed that both dimer (220 kDa) and monomer (140 kDa) forms of PSGL-1 were detectable (panel A, lane 1) and that isotype control antibody did not immunoprecipitate any detectable material (panel A, lane 2). Comparative densitometric scans of lanes 1 and 2 in panel A by means of NIH ImageJ software confirmed the appearance of 220- and 140-kDa forms of PSGL-1 immunoprecipitated from radiolabeled lysate (panel B, lane 1).