Fig. 6. Interaction between Dnmt3L and the Dnmt3 family methyltransferases. (A) COS cells transfected with GFP or GFP-3L alone (lanes 6 and 7) or along with myc-3a, myc-3b, or myc vector (lanes 1-5) were lysed and immunoprecipitated with anti-myc antibody. The bound proteins were separated on a 10% SDS-PAGE gel and immunoblotted with anti-GFP antibodies. The expression level of myc-3a and myc-3b in lanes 1 and 3 is shown by immunoblot using anti-myc antibody (lanes 8 and 9). The position of the GFP and GFP-3L is indicated. (B) NIH3T3 cells were transfected with myc-3L alone (not shown) or with myc-3L and GFP, GFP-3a, or GFP-3b. After 12 hours, the cells were fixed and immunostained with anti-c-myc antibodies followed by rhodamine-conjugated goat anti-mouse secondary antibodies. The myc-3L and GFP-3a proteins in transfected cells are shown as red and green fluorescence, respectively. The nuclei are stained with DAPI. The typical myc-3L localization patterns fall into four categories: nuclear, cytoplasmic, diffused and nuclear peripheral, and are shown in the top panels (red). In myc-3L alone or myc-3L and GFP, only cytoplasmic and diffused patterns were observed. In cells co-transfected with myc-3L and GFP-3a or GFP-3b, myc-3L is localized predominantly in the nuclei and colocalized with GFP-3a or GFP-3b. The percentage of cells expressing both myc-3L and GFP-3a or myc-3L and GFP-3b in each category is shown at the bottom. For myc-3L alone, 30 nuclei were scored and for other cotransfection experiments, 100-150 nuclei were scored.
