FIG 1.

Mutant p53 inhibits MDM2 E3 ligase activity. (a) MDM2 was cotransfected with p53 mutants in H1299 cells for 48 h. Protein expression was detected by Western blotting. *, the G245S mutant contained N-terminal FLAG tag. (b) MDM2 was cotransfected with p53 mutants and His₆-ubiquitin in H1299 cells for 48 h. MDM2 self-ubiquitination was analyzed by Ni²⁺-NTA pulldown and Western blotting. (c) Dose-dependent inhibition of MDM2 self-ubiquitination by mutant p53. (d) MDM2 self-ubiquitination was abrogated by point mutation (H457S) or deletion of the RING domain. (e) MDM2 ubiquitination of MDMX was inhibited by mutant p53. (f) Purified MDM2-p53 or MDM2-R175H mutant complex was incubated with charged E2. The release of ubiquitin from E2 was detected by Western blotting.