Fig. 4.

T203 O-GlcNAcylation is required for dendritic spine formation and soma size maintenance in cultured hippocampal neurons. A The recombinant lentivirus-mediated protein-replacement assay construct, pLEMPRA-MeCP2, was generated to knock down endogenous mMeCP2 and express Flag-tagged ectopic hMeCP2. B Schematic of the experimental design. C Representative images of mouse primary hippocampal neurons infected with indicated lentivirus at DIV 7; lentivirus FUGW-GFP is the negative control. Neurons were collected and fixed at DIV 14 for immunofluorescent staining with anti-PSD-95 antibody for measurement of dendritic spines. Green (GFP), soma and dendrites of LEMPRA lentivirus-positive neurons; red, PSD-95-positive dendritic spines were shown in; boxed areas, higher magnification of PSD-95 positive puncta (red) along secondary dendritic branches; scale bars 10 μm and 5 μm). D Area of soma of indicated lentivirus-positive neurons (mean ± SEM, one-way ANOVA followed by the Bonferroni test, **P < 0.01). E, F Linear density and area of PSD-95 puncta along the secondary dendritic branches in indicated lentivirus-positive neurons (mean ± SEM, one-way ANOVA followed by the Bonferroni test, **P < 0.01, ***P < 0.001).