biostudies-literatureAnny CAH2020 Mariee202500547https://www.ebi.ac.uk/biostudies/studies/S-EPMC12781144biostudies-literatureUnknown27(1)A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β-catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O-GlcNAcylated, or left unmodified. Using site-specific incorporation of the unnatural phenylselenocysteine, a β-catenin-EGFP fusion protein is engineered allowing selective installation of a S-GlcNAc moiety via oxidative elimination and thiol-Michael ligation. Additional β-catenin variants, phosphomimetic T41E-mCherry and wild-type-blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfuChembiochem : a European journal of chemical biologySimultaneous Visualization of Distinct Posttranslational Modification States of β-Catenin Using Genetic Code Expansion and Click Chemistry.PMC12781144847568Biot CHuvent ISchulz CAnny CABray FLefebvre TSpriet CNouaille SFaure RfalseSimultaneous Visualization of Distinct Posttranslational Modification States of β-Catenin Using Genetic Code Expansion and Click Chemistry.A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β-catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O-GlcNAcylated, or left unmodified. Using site-specific incorporation of the unnatural phenylselenocysteine, a β-catenin-EGFP fusion protein is engineered allowing selective installation of a S-GlcNAc moiety via oxidative elimination and thiol-Michael ligation. Additional β-catenin variants, phosphomimetic T41E-mCherry and wild-type-blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfu2026-01-01T00:00:00Z2026 Jan2026-06-06T12:43:04.406Z2026-05-30T03:11:16.505ZS-EPMC127811444138709410.1002/cbic.202500547