Figure 2

Dnmt1S localisation is independent of phosphorylation. A) Schematic representation of GFP-Dnmt1S phosphorylation mutants. B) Western blot of transfected Cos-7 cells probed with an antibody against the N-terminal domain of Dnmt1 [25] showing expression of GFP-Dnmt1s wild type (wt) and phosphorylation mutants S396A and S396D with the expected molecular mass. Weak bands at 190 kDa representing endogenous Dnmt1L are indicated. C) and D) localisation of GFP-Dnmt1S phosphorylation mutants in mouse somatic cells and preimplantation embryos, respectively. Both mutant proteins localise to the nucleus of somatic cells and to the cytoplasm of preimplantation embryos like wild type GFP-Dnmt1S (Fig. 1B and C). In D embryos were fixed and immunostained with an anti-GFP antibody detected with a red fluorescent secondary antibody. The upper row shows GFP fluorescence and the lower row shows the immunofluorescent signal (IF). The IF gradient from the periphery to the centre of some embryos is an optical artefact depending on signal intensity [15].