Interactions among MBD proteins.

<p>(<b>A</b>) Upper panel: schematic representation of methyl-cytosine binding domain (MBD) proteins. The numbers stand for amino acid coordinates. (MBD) methyl-cytosine binding domain, (TRD) transcriptional repression domain, (CxxC) cysteine rich domain. Lower panel: In vitro pull down experiments using purified strep (st) fused MeCP2 or MBD2 and GFP-labeled full-length (fl) MBD proteins immobilized to GFP-binding protein (GBP) bound sepharose beads. The proteins were extracted using 0.5 M NaCl containing lysis buffer. The interaction assays were performed either in PBS supplemented with 0.05% NP-40 (stMeCP2) or in PBS plus 150 mM NaCl and 0.05% NP-40 (MBD2st). Interacting st-taged fl MeCP2 and MBD2 were assessed by Western blot using st-HRP conjugate. Coomassie Brilliant Blue (CBB) staining of the SDS-gel after protein transfer shows GFP-labeled immobilized MBD proteins used for the pull-down assay. (<b>B, C, D</b>) Interactions between MeCP2 and MBD2 are not bridged by DNA. <i>In vitro</i> pull-down experiments were performed using immobilized RFP-fused MeCP2 (MeCP2R) or MBD2 (MBD2R) and GFP-labeled MeCP2 (MeCP2G) or MBD2 (MBD2G) either with or without addition of ethidium bromide (EtBr; 10 µg/ml). All proteins were extracted in 1 M NaCl containing lysis buffer. In the case of MeCP2 homo-interactions (<b>B</b>), the interaction was performed in PBS plus 0.05% NP-40 buffer. For the homo-interactions of MBD2 (<b>C</b>), PBS was additionally supplemented with 110 mM NaCl and 0.05% NP-40, and for the hetero-binding of MBD2 and MeCP2 (<b>D</b>), PBS plus 125 mM NaCl and 0.05% NP-40 was used. (<b>B, C, D</b>) For input control (I), ¼ of the protein amount used for the interaction assay of the immobilized RFP-tagged proteins was loaded on SDS-PAGE and stained with CBB. Also ¼ of the GFP-tagged proteins used for the pull-down were visualized by western blot using anti GFP (I). Interacting GFP-fused MeCP2 or MBD2 (B) were assessed by western blot using anti GFP antibody.