Fig 5.

Stimulation by DNMT3L is detected at the luciferase gene integrated in the genome. Two cell lines (O13 and O21) carrying the luciferase gene at distinct loci were transfected with Dnmt3a or Dnmt3a + DNMT3L expression vectors. After transfection (3.5 days), genomic DNA was harvested and digested with excess HindIII and HhaI, a methylation-sensitive enzyme. The resulting DNA species were then separated by agarose gel electrophoresis, Southern transferred onto a membrane, and hybridized to a probe complementary to the luciferase gene. The presence of Dnmt3a and/or DNMT3L expression vectors was as indicated. CpG methylation prevents cleavage by HhaI and results in higher molecular weight species that can be easily distinguished.