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. 2003 Apr;23(8):2834–2843. doi: 10.1128/MCB.23.8.2834-2843.2003

FIG. 2.

FIG. 2.

MBD1 interacts with a transcriptional modulator, MCAF. (A) Structure of MBD1. MBD1 contains an MBD, three cysteine-rich CXXC domains, and a TRD. A yeast two-hybrid screen using the TRD identified MCAF. Two amino acid residues within the TRD (indicated by capital letters) are important for MBD1-dependent repression (Fig. 4). (B) Expression of MCAF. Anti-MCAF polyclonal antibodies and a vector expressing FLAG-tagged MCAF were utilized. (C) Complex formation of MBD1 and MCAF. Endogenous MCAF and HA-tagged MBD1 were immunoprecipitated from HeLa cells. IP, immunoprecipitation; Ab, antibody. (D) Interaction between endogenous MBD1 and MCAF. Mouse anti-MBD1 monoclonal antibodies and rabbit anti-MCAF polyclonal antibodies were used for immunoprecipitation. (E) Biochemical binding assay. MCAF possesses two distinct conserved domains, named domain 1 (Δ18) and domain 2 (Δ8). GST and GST-MCAF (2 μg) were immobilized on glutathione-agarose beads and incubated with His-tagged TRD of MBD1 (2 μg). The input indicates 10% of the indicated proteins in the reaction mixture.