FIG. 4.

Overexpression of MDMX does not change p21 ubiquitylation in cells. (A and B) Coexpression of MDMX with p21 does not change the p21 ubiquitylation pattern in cells. p53^−/− MDMX^−/− MEF cells (A) or H1299 cells (B) were transfected with a combination of plasmids encoding His₆-ubiquitin (His-Ub), human p21, p21-6KR, or c-myc-MDMX, as indicated. The cells were treated with 10 μM MG132 for 6 h prior to harvest. Cell lysates were incubated with Ni-NTA beads and washed intensively for SDS-PAGE and WB analyses using antibodies against p21 or MDMX. The cell lysates (50 μg/sample) were used for straight WB, as indicated. (C) MDMX mediates the degradation of the ubiquitylation-deficient p21 mutant independent of MDM2. p53^−/− MDM2^−/− MEF cells were transiently transfected with plasmids encoding p21-6KR or GFP-MDMX. The transfected cells were harvested 48 h posttransfection and lysed. The cell lysates (70 μg/sample) were subjected to SDS-PAGE, followed by WB, as indicated.