[¹⁴C]4F-GalNAc distribution in culture media and glycoproteins. Cell culture media containing [¹⁴C]4F-GalNAc (0.5 μCi/mL, 100μM) was placed under standard growth conditions either in the presence or absence of HL-60 cells for 38 hours. Growth media and cells were harvested separately. (A-B) Culture media harvested from cultures with (A) and without HL-60 (B) separated on Biogel P-2 column. (C) Samples at the void volume (samples 1 and 4), peak fractions in panel A (samples 2 and 3) and panel B (sample 5) were separated using TLC with chloroform/methanol (5:1) as mobile phase. Phosphorimaging detected radioactivity on TLC plates. Nonradiolabeled, nonacetylated 4F-GalNAc (lane 6) and per-acetylated 4F-GalNAc (lane 7) standards were run in parallel and developed using ethanol/sulfuric acid spray followed by charring. Addition of acetylated 4F-GalNAc to HL-60 cells leads to deacetylation as evidenced by prominent peak 3 in the chromatogram. (D) PSGL-1 was immunoprecipitated from HL-60 lysates cultured with [¹⁴C]4F-GalNAc using Ab H-300. This immunoprecipitate and PSGL-1–depleted cell lysate were resolved on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and blotted onto nitrocellulose membrane. Radioactivity was measured using scintillation counter in panels A and B; and using phosphorimaging in panels C and D. 4F-GalNAc is incorporated into leukocyte PSGL-1 and other glycoproteins.