Fig 2: The NLS of MeCP2 Interacts with KPNA3 and KPNA4(A) Schematic of the primary structure of MeCP2 showing the positions of the methyl-CpG binding domain (MBD), the first AT-hook (hook 1), the nuclear localization signal (NLS), and the NCOR1/NCOR2 interaction domain (NID). The expanded view of the NLS depicts the two basic patches of this motif (gray), the overlapping AT-hook 2 (hook 2) (pink), and the positions of the RTT-causing truncation mutations R255X, R270X, and G273X (bold). Horizontal black lines indicate the extent of the deletions used in the MBD and in AT-hook 1.(B) Immunoprecipitation (IP) of MeCP2, Kpna3, and Kpna4 from extracts of wild-type (WT) mouse brain using an antibody against MeCP2. IP from Mecp2 knockout (KO) brain is a negative control.(C) Coomassie stain of purified GST-MeCP2NLS (GST-NLS) and GST alone.(D) Western blotting reveals in vitro-translated 3×FLAG-tagged Kpna4 is bound by GST-NLS, but not by GST alone.

Fig 3: NLS Mutation Does Not Affect the Expression Level or Localization of MeCP2G273X-EGFP in Mouse Brain(A) Western blotting and quantification of MeCP2 levels in WT, Mecp2-EGFP (EGFP), Mecp2G273X-EGFP (G273X), and Mecp2G273XΔNLS-EGFP (NLS) whole-mouse brain extracts (n = 4). Loading control is anti-histone H3. Quantification is presented as means ± SD.(B) IP of MeCP2-EGFP from extracts of brains from animals as in (A) using an antibody against EGFP. MeCP2 as well as bound Kpna3 and Kpna4 are detected by western blotting.(C) Fluorescence microscopy of MeCP2-EGFP in CA1 layer of hippocampus in brain sections of Mecp2G273X-EGFP (G273X), and Mecp2G273GXΔNLS-EGFP (NLS) mice. The scale bars represent 10 μm.(D) Western blot of MeCP2 levels in cytoplasmic (C) and whole-cell (W) fraction from WT, Mecp2-EGFP (EGFP), Mecp2G273X-EGFP (G273X), and Mecp2G273GXΔNLS-EGFP (NLS) mouse brain. Control cytoplasmic protein is gamma-tubulin.