FIG. 6.

MBD2 methylation controls the affinity of MBD2 for methyl-CG DNA. (A) FLAG-MBD2 was expressed in human (293T) cells and purified as described in the legend to Fig. 3B, except Empigen BB replaced Triton X-100 in the buffer. Biotinylated double-stranded DNA (40-mer containing 12 CGs, either unmethylated or 5-methylcytosine methylated on both strands) was immobilized on streptavidin-agarose. FLAG-MBD2 was incubated with the immobilized DNA in the presence of 0.25 mg/ml tRNA for 2.5 h. The unbound fraction was collected, the resin was washed in buffer containing 350 mM NaCl, and the bound fraction was detached from the resin by boiling in SDS sample buffer. Unbound and bound fractions were analyzed by SDS-PAGE, Coomassie blue staining, and Western blotting. Input represents FLAG-MBD2 added to the immobilized DNA. (B) FLAG-MBD2 was prepared and incubated with immobilized methyl-CG DNA as described for panel A. The resin was then step eluted with increasing concentrations of NaCl as shown. Unbound, bound, and eluted fractions were analyzed as described for panel A. The arginine-methylated population of MBD2 was detected by Western blotting with ASYM24 and SYM10 antibodies.