FIGURE 5.

p53 ubiquitination mainly takes place in the cytoplasm where Mdm2 and MdmX colocalize. A, in vivo ubiquitination of endogenous p53 in 293T cells. 293T cells were transfected with 200 or 400 ng of pcDNA3.0-HA-MdmX DNA per 60-mm plate in the absence (lanes 1–3 and 7–8) or presence of pFLAG-CMV-Mdm2 (400 ng DNA/60-mm plate). Twenty-four hours later, cytoplasmic and nuclear fractions were prepared, and Western blot analysis of p53 (using a mixture of DO-1 and Pab1801 antibodies), Mdm2 (using a mixture of 2A9 and 4B11 antibodies), and MdmX (anti-HA) was performed. PCAF and tubulin were used as loading controls as well as nuclear and cytoplasmic markers, respectively. L expo and S expo indicate long and short exposure, respectively. The asterisk indicates the position of endogenous human Mdm2. B, in vivo ubiquitination of endogenous p53 in U2OS cells. U2OS cells were treated with a nuclear export inhibitor, leptomycin B (LMB; 20 ng/ml) for various times; cytoplasmic and nuclear fractions were prepared and subjected to Western blot analysis. The arrowhead indicates a short fragment of endogenous Mdm2.