Figure 5.

Fluorescence lifetime imaging microscopy-fӧrster resonance energy transfer (FLIM-FRET) to monitor substrate-specific OGT activity in live cells. An OGT substrate fused with fluorescent protein can be metabolically labeled by azido sugar in cells. Click-chemistry conjugation of glycosylated protein to an acceptor fluorophore leads to proximity-induced FRET between donor and acceptor on the glycosylated substrate. The reduced fluorescence lifetime of the donor can report the O-GlcNAcylation on the protein substrate.