Fig. 4.

Cell-based validation of ChREBP O-GlcNAcylation. A, OGT overexpressed Hepa1–6 cells were exposed to indicated conditions with 200 μm Ac₃6AzGlcNAc for 24 h. Lysates were assessed for OGT, α-Tubulin, and Eluates for the biotin-modified protein pools by WB. B, Hepa1–6 cells were cotransfected with OGT and ChREBP and exposed to indicated conditions with 200 μm Ac₃6AzGlcNAc. Eluates from the biotin pull-down assay were assessed for ChREBP, p300, OGA, OGT, Mlx and SP1by WB. All these proteins and α-Tubulin from cell lysates were immunoblotted as references. C, Metabolic labeled cell lysates were reacted with PEG mass-tag and assessed for ChREBP (up) and SP1 (down) respectively by WB. All data are representative of 3 independent experiments.