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. 2017 Apr 17;292(22):9150–9163. doi: 10.1074/jbc.M116.766568

Figure 3.

Figure 3.

O-GlcNAcylation of p65 at T305 enhances NF-κB transcriptional activity. A, NF-κB p65-deficient MEF cells were transfected with plasmids expressing IκBα and 3xFLAG-tagged WT p65, T305A, S319A, S337A, T352A, S374A, or vector and plasmids expressing HA-OGT or lacZ together with NF-κB luciferase and Renilla luciferase reporter vectors. The luciferase activity relative to WT p65 was measured 24 h later and normalized with the Renilla activity. B, HEK 293T cells were transfected with plasmids expressing 3xFLAG-tagged WT p65, T305A, S319A, T305A/S319A, or vector and plasmids expressing HA-OGT or lacZ together with NF-κB luciferase and Renilla luciferase reporter vectors. The luciferase activity relative to WT p65 was measured 24 h later and normalized with the Renilla activity. C, plasmids expressing 3xFLAG-tagged WT p65, T305A, T352A, or T305A/T352A and the HA-OGT plasmid were transfected into HEK 293T cells. FLAG-tagged p65 was immunoprecipitated, and O-GlcNAc and p65 were detected by Western blotting. D, NF-κB p65-deficient MEF cells were transfected with plasmids expressing IκBα and 3xFLAG-tagged WT p65, T305A, T352A, T305A/T352A, or vector and plasmids expressing HA-OGT or lacZ together with NF-κB luciferase and Renilla luciferase reporter vectors. The luciferase activity relative to WT p65 was measured 24 h later and normalized with the Renilla activity. Data represent the means ± S.D. (error bars) from at least three independent experiments. * and #, p < 0.05; ** and ##, p < 0.01; *** and ###, p < 0.001.