Fig. 3.

PuPhgt is a GlcNAc-transferase. (A) SDS-PAGE gel of purified FLAG-PuPhgt, after staining with Coomassie Blue, or after western blotting with mAb M2 (anti-FLAG). (B) Sugar nucleotide hydrolysis. Purified PuPhgt was incubated with the indicated sugar nucleotide and, after 17 h, the reaction was quenched and the concentration of UDP generated was measured using the UDP-Glo assay. (C) GlcNAc-transferase assays using DdSkp1 peptides. A total of 1 mM Pro-peptide(133–155) or Hyp-peptide(133–155) was incubated with PuPhgt or DdGnt1 in the presence of 1 μM UDP-[³H] GlcNAc and absence of αKG. Incorporation was measured by liquid scintillation counting of the peptide band excised from an SDS-PAGE gel. Error bars represent standard deviation of three technical replicates of a single trial; similar results were obtained using a separate enzyme preparation.