Figure 7.

LPS-induced NF-κB-p65 subunit and IKKβ O-GlcNAcylation and subsequent NF-κB signalling activation in Jurkat cells were blocked by BtGH84 and AkkGH84. Jurkat cells were pretreated with BtGH84, AkkGH84, BtGH84-2D or AkkGH84-2D, followed by LPS incubation for. (A) Whole cell extracts were analysed with immunoblots for O-GlcNAc, NF-κB-p65, IKKβ and IκBα. β-actin serves as a loading control. (B) O-GlcNAcylated proteins in Jurkat were pulled down using sWGA beads. O-GlcNAc, NF-κB-p65, IKKβ and IκBα in the pull-down complexes were detected using immunoblots. (C) Whole cell extracts of Jurkat were immunoprecipiated with anti-NF-κB-p65 or anti-IKKβ antibody. The O-GlcNAcylated NF-κB-p65 and IKKβ were detected using anti-O-GlcNAc antibody. (D–F) Cytosolic and nuclear sections were analysed by immunoblots for IκBα and NF-κB-p65, respectively. β-actin serves as a loading control of cytosolic section; histone-H3 serves as a loading control of nuclear. (G) Jurkat were transfected with pGL3/NF-κB and PRL, followed by treated with BtGH84, AkkGH84, BtGH84-2D or AkkGH84-2D as described above. Afterward, cells were harvested for luciferase activity assay. (H, I) Proinflammatory cytokines levels in treated Jurkat. Data represent the mean of three repeats per group with the SD; *P<0.05, **P<0.01, ***P<0.001. LPS, lipopolysaccharide; NS, not significant; sWGA, succinylated wheat germ agglutinin agarose.