Fig. 4.

Ac₄GalNAz treatment results in GALE-dependent UDP-GlcNAz biosynthesis and labeling of O-GlcNAcylated proteins. (A) CHO or ldlD CHO cells were treated with vehicle or Ac₄GalNAz for 24 h. Nuclear and cytoplasmic extracts were prepared, reacted with phosphine-Flag, and analyzed by immunoblot. Total protein was visualized by India ink staining. (B) CHO or ldlD mutant CHO cells were treated with vehicle or Ac₄GalNAz for 72 h. Cells were fixed and azidoglycans detected via reaction with phosphine-Flag and immunofluorescence microscopy using an anti-Flag-FITC antibody conjugate. A Hoechst 33258 stain was included to visualize nuclei. (Top) FITC only. (Bottom): FITC/Hoechst merge.