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. Author manuscript; available in PMC: 2012 Oct 6.
Published in final edited form as: Neuron. 2011 Oct 6;72(1):72–85. doi: 10.1016/j.neuron.2011.08.022

Figure 1. Selective Loss of the Activity-Dependent Phosphorylation of MeCP2 S421 in vivo Preserves the Expression Level and Localization of MeCP2.

Figure 1

(A) Western blot analysis of nuclear extracts prepared from the forebrains of two 4-week-old male MeCP2 S421A knock-in mice (S421A KI) and their wild-type (WT) littermate using antibodies specific to MeCP2 pS421, and total MeCP2. Parrallel blots for pS133 CREB and total CREB show that there is no gross disruption of activity-dependent signaling in the brain.

(B) Anti-total MeCP2 Western blots of lysates from E16 + 5 DIV dissociated hippocampal neurons derived from MeCP2 S421A knock-in mice or wild-type littermates. (i) Arrow indicates the S421 phosphorylation-dependent slowly-migrating form of MeCP2 induced by 60 minute 55 mM KCl stimulation in wild-type cells (ii) 30 minute pre-treatment with okadaic acid (0.25 μM) to inhibit protein phosphatase activity increases the proportion of slowly-migrating MeCP2 induced by the 60 minute membrane depolarization. MeCP2 S421A neurons do not display this slowly-migrating form of MeCP2 in response to membrane depolarization.

(C) Quantification of Western blot analysis for the forebrains of 4-week-old MeCP2 S421A mice (n=3) and their wild-type littermates (n=3) using anti-total MeCP2 antiserum. Data are mean ± SEM. See also Figure S1.

(D) Immunocytochemistry of E16 + 22 DIV cortical neurons derived from MeCP2 S421A mice and wild-type littermates using an antibody against total MeCP2 and costaining with anti-histone H3 lysine 18 acetylation (H3K18Ac, active chromatin), anti-histone H3 lysine 9 trimethylation (H3K9me3, heterochromatin) or anti-histone H3 lysine 27 trimethylation (H3K27me3, repressed chromatin). Single 63X confocal planes of representative neuronal nuclei are shown.