FIGURE 5.

OGT F460A is a dominant negative mutant protein used in the NCM hyperglycemia model for decreasing Ogg1 O-GlcNAcylation. A, construction of the OGT dominant negative mutant F460A. The OGT protein is comprised of two domains: the N-terminal TPR domain, which contains a variable number of TPR repeats, and the C-terminal catalytic domain containing two conserved regions (CD 1 and CD 2). The F460A mutation was in the CD 1 region of the OGT catalytic domain, as indicated in red. B, total protein O-GlcNAcylation from NCM cultured in either NG or HG and transduced with either Adv-ctr or Adv-OGT F460A. OGT and actin levels are also reported. MW, molecular weight; A.U., arbitrary units. C, co-IP experiments showing reduced Ogg1-specific O-GlcNAcylation and increased interaction between Ogg1 and OGT F460A. IP signals were normalized to input levels, except for RL2 IP detection, which was normalized to Ogg1 detected in the immunoprecipitate. Actin was used as a loading control. All data are expressed as mean ± S.E., represent at least five independent experiments, and were analyzed by comparing all conditions versus NG Adv-ctr. *, p < 0.05; **, p < 0.01.