cMBD2 affinity copurifies the other components of the MeCP1 complex. (A) Western blot analysis of cMBD2 and streptavidin expression in 6C2 cells containing the BAP-cMBD2 construct with or without the biotin ligase BirA. Equal expression of cMBD2 is seen in BirA^- and BirA⁺ BAP-cMBD2 6C2 cells, whereas biotinylation of cMBD2 only occurs in the BirA⁺ cells. (B) Western blot analysis of the eluate from cMBD2 pull-down experiments in BAP-cMBD2 6C2 cells. Abundant biotinylated cMBD2 as well as the major components of the MeCP1 complex are seen in the eluate from pull-down assays performed using BirA⁺ BAP-cMBD2 6C2 cells as the input. In contrast, no biotinylated cMBD2 or any MeCP1 components are seen in the eluate from pull-down assays performed using BirA^- BAP-cMBD2 6C2 cells as the input. (C) Sypro Ruby-stained protein gel of the eluate from cMBD2 pull-down experiments in BAP-cMBD2 6C2 cells. Interestingly, despite equal amounts of input protein, less protein elutes from streptavidin beads bound to BirA⁺ BAP-cMBD2 6C2 cell nuclear extract. The identities of bands in the gel were determined by tandem-mass spectrometry on excised and trypsin-digested bands. The identities of the indicated bands are listed on the right. At least 2 experimentally derived peptides were required for protein identification.