FIG. 7.
MDM2-mediated p53 ubiquitination occurs preferentially in the cytoplasm. (A to C) Wild-type and mutant MDM2s were cotransfected with plasmid DNA encoding wild-type p53 (A), nuclear import mutant p53KRKKK (B), and nuclear export mutant p53NES (C) into U2-OS cells. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 6 h and then harvested in SDS lysis buffer. The cell lysate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Different portions of the membrane were blotted with antibodies to MDM2 (SMP14), p53 (DO1), and β-actin, as indicated. (D) H1299 cells were transiently transfected with plasmid DNA encoding wild-type MDM2, wild-type p53, and various p53 mutants, as indicated. Twenty-four hours after transfection, cells were lysed with 0.5% NP-40 lysis buffer. The cell lysate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Different portions of the membrane were blotted with antibodies to MDM2 (SMP14) and p53 (DO1), as indicated.