Fig. 3.

In vitro cell-free validation of ChREBP O-GlcNAcylation. A, Schematic diagram of the method. B, Structures of tool molecules. C, Detection of O-GlcNAcylated ChREBP. Reaction mixture prior to pull down (Input) and the captured proteins (Elution) were immunoblotted with antibodies toward ChREBP and OGT. UDP-GlcNAc addition and OGT administration strongly enhanced the modification (lane 2, 5). Control experiments in absence of Y289L GalT1 (lane 3) or biotin derivate (lane 4) demonstrated selective labeling. All data are representative of 3 independent experiments.