FIGURE 4.

The TET3-OGT complex is an epigenetic activator specific for GnT-IX gene. A, Neuro2A cells were transfected with the TET3-3×FLAG expression vector or the empty vector (Mock). The cell lysates (Input) were subjected to IP with anti-OGT or anti-FLAG antibody and then Western blotted with an anti-OGT or anti-FLAG antibody. B, Neuro2A cells were treated with control siRNA (siCont) or TET3 siRNA for 24 h. Total RNAs were extracted, and the mRNA levels of TET3 were quantified and normalized to those of rRNA. The mRNA/rRNA level of TET3 knockdown sample relative to that of control sample is shown (n = 3, *, p < 0.05, Student's t test). C, Neuro2A cells were treated with control siRNA or TET3 siRNA for 24 h, and then ChIP assays were performed with anti-OGT or anti-O-GlcNAc (RL2) antibody. Genomic regions around the transcriptional start sites of GnT-III, -V, and -IX and Fut8 genes were analyzed by real-time PCR. The amounts of DNA relative to those with control IgG are shown (left, n = 5; right, n = 3, *, p < 0.05, Student's t test). D, Neuro2A cells were treated with control siRNA or OGT siRNA, and the cell lysates were Western blotted with an anti-OGT, anti-actin, or anti-O-GlcNAc antibody. E, Neuro2A cells were treated with control siRNA or OGT siRNA for 24 h. Total RNAs were extracted, and the mRNA levels of GnT-III, -V, and -IX and Fut8 were quantified and normalized to those of rRNA. The mRNA/rRNA levels of OGT knockdown samples relative to those of control samples are shown (n = 4, *, p < 0.05, **, p < 0.01, Student's t test). All graphs show means ± S.E.