Fig. 3.
GlcNAz does not transit the UDP-GlcNAc pyrophosphorylase step in the GlcNAc salvage pathway. (A) Jurkat or 293T cells were treated with vehicle or 100 μM azidosugar for 24 h. Ethanol extracts were made and analyzed by HPAEC. Synthetic UDP-GlcNAz and UDP-GalNAz standards were included. Asterisk: an unknown species present in all cell-derived samples. (B) 293T cells were mock-transfected or transfected with the expression construct indicated and treated with vehicle or Ac4GlcNAz for 24 h. Cell lysates were reacted with phosphine-Flag and analyzed by immunoblot. (C) 293T cells were mock-transfected or transfected with an AGX2-myc expression construct. After 21 h, all cells were treated with 100 μM Ac4GlcNAz, samples were harvested by ethanol extraction after 3, 6, or 9 additional hours and analyzed by HPAEC. Asterisk: an unknown species present in all cell-derived samples. (D) Ethanol-insoluble protein fractions from the samples in C were resuspended in 8 M urea, reacted with phosphine-Flag, and analyzed by immunoblot.