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. 2006 Dec 14;26(1):102–112. doi: 10.1038/sj.emboj.7601469

Figure 1.

Figure 1

C-terminal tail of MDM2 is required for MDM2-mediated p53 degradation and ubiquitylation. (A) C-terminal tail sequences of MDM2 proteins were aligned using BOXSHADE 3.21 software at http://www.ch.embnet.org/software/BOX_form.html. (B) MDM2 C-terminal deletions are not able to target p53 for degradation. U2OS cells were transiently cotransfected with FLAG-p53, GFP and MDM2 C-terminal deletions and analyzed by Western blotting. (C) MDM2 C-terminal tail deletions prevent efficient p53 ubiquitylation in vitro. Bacterially expressed wild-type and mutant GST-MDM2, in vitro-translated p53, recombinant ubiquitin, E1 and E2 enzymes were incubated in the reaction buffer at 37°C for 2 h. A negative control reaction was prepared without the E2 enzyme. Reaction products were resolved by SDS–PAGE and analyzed by Western blotting with anti-p53 DO-1. (D) MDM2 C-terminal deletions prevent efficient p53 ubiquitylation in vivo. U2OS cells, transiently cotransfected with FLAG-p53 (0.2 μg) and MDM2 C-terminal deletion mutants (1.5 μg), were treated with 10 μM MG132 24 h after transfection and analyzed by Western blotting.