Figure 3.

More Mdm2 protein colocalizes with p19^ARF in the nucleolus when Mdm2 shuttling is allowed for a longer period of time. HeLa cells were cotransfected with plasmids encoding Mdm2 and p19^ARF. Thirty-six hours after cotransfection, the heterokaryon assay was performed, in which cells were incubated for 5 hr at 37°C to allow protein shuttling after cell fusion. The red signal represents the Mdm2 protein (a), and p19^ARF is represented by the green signal (b). The green signal observed in the mouse nucleus (b) represents the endogenous p19^ARF in mouse 2KO cells. The nuclear staining by DAPI is represented by the blue color (d). Phase-contrast optics were used to confirm the fusion of cells (e). Under this experimental condition, Mdm2 shows a much higher level of colocalization with p19^ARF in the nucleolus (compare a-c to Fig. 2B). In addition, nucleo-cytoplasmic shuttling of Mdm2 was blocked by p19^ARF (a).