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. 2018 Jul 16;115(31):8019–8024. doi: 10.1073/pnas.1805538115

Fig. 5.

Fig. 5.

O-GlcNAcylation attenuates JUN phosphorylation and JUN/AP-1 transcription. (A and B) An antagonistic relationship in JUN between O-GlcNAcylation and phosphorylation. (A) Western blot analysis of immunoprecipitated FLAG-tagged wild-type JUN in HEK293 cells treated with 1 µM Thiamet-G (TG) and/or overexpressing OGT as indicated. (B) Western blot analysis of immunoprecipitated FLAG-JUN wild-type (WT), dephosphorylation mimetic (4A: S63A, S73A, T91A, T93A), or phosphorylation mimetic (4D: S63D, S73D, T91D, T93D) in HEK293T cells expressing OGT and treated with Thiamet-G. Numeric values denote quantification of O-GlcNAc RL2 normalized by total FLAG-JUN. (C) O-GlcNAcylation negatively affects AP1 transcriptional activity. Luciferase reporter assay using construct containing 3× AP-1 binding sites in HEK 293T cells expressing OGT and/or treated with TG as indicated. RLU, relative luciferase unit. **P < 0.01, triplicate; ***P < 0.001, triplicate. (D) Ogt knockdown enhances AP-1 transcription and JUN transactivation in HEK293T cells. Scrambled shRNA (sh-Ctrl), Ogt shRNA (sh-OGT) with or without JUN overexpression (JUN). ***P < 0.001.