Dnmt3a2:R878H antagonizes WT Dnmt3a and Dnmt3b. (A) Myc-tagged Dnmt3a2 or Dnmt3b2 and Flag-tagged Dnmt3a2:RH or GFP (control) were expressed simultaneously in 7aabb cells (passage number: ∼80), and stable clones were analyzed by immunoblotting with anti-Flag, anti-Myc, and anti–β-actin antibodies and by Southern blot for methylation of MSRs. J1 and untransfected 7aabb ES cells were used as controls. (B-C) Myc-tagged Dnmt3a2 or Dnmt3a2:RH was transfected into (B) 8bb or (C) 6aa ES cells, and stable clones were analyzed by immunoblotting with anti-Myc and anti–β-actin antibodies and by Southern blot for methylation of MSRs. 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 of representative samples (supplemental Figure 4). NT, not tested. To avoid the effect of culturing time on DNA methylation, stable clones expressing the control and R878H proteins were generated from same batches of parental cells and were cultured for the same periods of time (in most cases, 7-10 days for generating stable clones and 7-10 days for expansion) before genomic DNA was isolated.