Fig. 2

Nuclear localization, DNA binding affinity and transcriptional activity of O-linked N-acetylglucosamine glycosylated (O-GlcNAcylated) p65. a We confirmed that O-GlcNAcylation of p65 was enhanced by ThiaMet-G in MH7A cells using western blotting immunoprecipitation with both anti-p65 and anti-O-GlcNAc. b Western blotting (WB) was performed using anti-p65 and anti-O-GlcNAc and succinylated wheat germ agglutinin (sWGA)-affinity purification were performed for precipitating the O-GlcNAcylated proteins. ThiaMet-G significantly increased the nuclear translocation of p65 in TNF-α-simulated MH7A cells within 1 h. c and d Avidin-biotin complex DNA (ABCD) lysis buffer was used for MH7A cells lysis to measure DNA binding affinity. Lysates were incubated with biotinylated nuclear factor-kappa B (NF-κB) promoter site DNA. NF-κB transcriptional activity was measured in MH7A cells transfected with NF-κB-dependent luciferase reporter gene construct 4xkB- Luc. TNF-α-simulated MH7A cells exhibited significant increases in both the DNA binding affinity and transcriptional activity of p65, compared to controls and these effects were further enhanced following treatment with ThiaMet-G. e TNF-α alone augmented the level of O-GlcNAcylated p65 in fibroblast-like synoviocyte lysates. (*P <0.05)