Fig. 2.

OGT directly interacts with and O-GlcNAcylates MeCP2. A Western blots of mouse brain lysates immunoprecipitated with anti-MeCP2 antibody, followed by Western blots with an anti-OGT antibody. IB, immunoblotting. MW, molecular weight. B Reciprocal co-IP assay of mouse brain lysates with an anti-OGT antibody, followed by Western blots with an anti-MeCP2 antibody. C, D GST pull-down assays for His-hMeCP2 and GST-OGT, or His-OGT and GST-hMeCP2. Input and pull-down samples were analyzed with anti-MeCP2 and anti-OGT antibodies. Input represents 5% of the amount used for pull-down assay. E, F Lysates of HEK293T cells transfected with HA-tagged hMeCP2 and GFP-tagged full-length and indicated deletion mutants of OGT immunoprecipitated with anti-HA or anti-GFP antibodies, followed by Western blots with anti-GFP and anti-HA antibodies, respectively. Input represents 5% of the amount used for pull-down assay. G, H Representative quantification of Western blot results followed by co-IP assay in E and F, respectively. Histograms show the mean ± SEM. One-way ANOVA followed by the Bonferroni test, **P < 0.01. I Summary diagram of the serial deletion mutants of OGT and their binding capacities to MeCP2. Deletion of either the entire TPRs or TPRs 1–3 within OGT dramatically disrupts its binding to MeCP2.