Fig. 6

Ogt catalyzes O-GlcNAcylation of NF-κB on S384 and inhibits the activation of NF-κB signaling pathway. a, b Reciprocal IP-WB analysis showed that Ogt readily precipitated p65 (a), and vice versa in astrocytes (b). c, d Reciprocal IP-WB analysis showed that O-GlcNAcylation precipitated p65 (c), and vice versa in astrocytes (d). e Click-iT reaction results revealed that p65 was modified with O-GlcNAcylation. f–h WB assay showed that under Ogt knock down (KD) significantly decreased the levels of Ogt and O-GlcNAcylation, but significantly increased the level of p-p65 in N2a cells (f, Input part). IP with HA (HA-p65) antibody followed by WB assays (f, IP part) showed that O-GlcNAcylation levels of exogenous p65 (g) were significantly decreased, and the level of p-p65 (h) was significantly increased under Ogt KD condition. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. i–k WB assay showed that ectopic expression of Ogt (Ogt-Flag, OE-Ogt) and significantly increased the levels of Ogt and O-GlcNAcylation without affecting the levels of total p65 and p-p65 in N2a cells (i, Input part). IP with HA (HA-p65) antibody followed by WB assays (i) showed that the O-GlcNAcylation level of p65 (p65-HA) (j, IP) significantly increased, but the level of p-p65 (k) significantly decreased. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. l–n IP followed by WB assays showed that Ogt KD promoted the binding of Gsk3β to p-65. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o–q IP followed by WB assays showed that mutation of S384A significantly increased the binding of Gsk3β to p-65 compared to other O-GlcNAcylation sites. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(4, 10)} = 47.49 for p, F_{(4, 10)} = 42.31 for q