Fig. 4.

PuPhgt preferentially modifies PuSkp1A relative to PuSkp1B in vitro. (A) The native protein sequences of PuSkp1A and PuSkp1B were expressed in E. coli, purified by conventional chromatographic methods and analyzed by SDS-PAGE with staining by Coomassie blue for total protein. (B) PuPhgt, supplemented as indicated with DdPhyA or DdGnt1, was incubated in the presence of atmospheric O₂ and αKG with the indicated type of 1.8 μM Skp1 and 2 μM UDP-[³H] GlcNAc for 2 h. Incorporation of radioactivity into Skp1 was determined after precipitation with trichloroacetic acid. (C) Similarly, a mixture of DdPhyA and DdGnt1 was incubated with the indicated type of Skp1. Error bars represent standard deviation of two technical replicates of a single trial; similar results were obtained using separate protein preparations.