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. 2008 Nov 6;105(45):17345–17350. doi: 10.1073/pnas.0806198105

Fig. 4.

Fig. 4.

O-GlcNAcylation of NFκB p65 occurs at Thr-322 and Thr-352. O-GlcNAcylation of NFκB p65 C-terminal deletion mutant proteins and Ser or Thr to Ala substitution mutant proteins. NFκB p65 KO MEFs were cotransfected with the vector alone or plasmids encoding the His-NFκB p65 deletion (lanes 1–12) or substitution mutants (lanes 13–36), along with the plasmid encoding FLAG-tagged OGT for 12 h. His-NFκB p65 immunoprecipitates (2nd row) were obtained from cellular extracts by using the anti-NFκB p65 polyclonal antibody and were analyzed by immunoblotting for O-GlcNAc (3rd row). Total levels of His-NFκB p65 mutants in these extracts were examined by using the anti-NFκB p65 monoclonal antibody (1st row), and the specificity of O-GlcNAcylation on NFκB p65 mutants was confirmed by decreasing the modification via the addition of 10 mM GlcNAc (4th row).