The R878 mutations severely impair the ability of Dnmt3a to methylate DNA but retain its ability to interact with wild-type Dnmt3a and Dnmt3b. (A) Myc-tagged WT Dnmt3a/3a2 or R878 mutant (R878H [RH], R878C [RC], or R878S [RS]) was transfected into 7aabb ES cells (passage number: ∼80), and stable clones were obtained. The cell lysates were analyzed by immunoblotting with anti-Myc and anti–β-actin antibodies, and genomic DNA was digested with MaeII and analyzed by Southern hybridization with a probe specific for the MSRs. Untransfected 7aabb cells and WT (J1) ES cells were used as controls. Densitometry was used to determine the relative methylation levels (methylation scores), as described in supplemental Figure 3, and bisulfite sequencing was used to quantify methylation levels in representative samples (supplemental Figure 4). NT, not tested. (B) Flag-tagged and Myc-tagged WT and/or mutant Dnmt3a2 (as indicated) were cotransfected in COS-7 cells, the cell lysates were immunoprecipitated with anti-Myc antibody, and the precipitated proteins (Myc IP), as well as total cell lysates (TCLs), were analyzed by immunoblotting with anti-Flag and anti-Myc antibodies. (C) Similar experiments as in B except that Flag-Dnmt3b1 or -Dnmt3b2 was used instead of Flag-Dnmt3a2. The Flag- and Myc-tagged proteins and the immunoglobulin G (IgG) heavy chain are indicated.