FIG. 6.

Overexpression of hHR23 in the presence of Mdm2 results in the accumulation of ubiquitylated forms of p53. (A) H1299 cells (p53 null cells) were transfected with expression constructs for p53, Mdm2, Flag-tagged hHR23, and His-tagged ubiquitin (for further details, see Materials and Methods). Twenty-four hours after transfection, cells were harvested and whole-cell lysates were prepared from an aliquot of the cells. Using the whole-cell lysate, expression levels of p53, Mdm2, and Flag-tagged hHR23 were determined by Western blot analysis with antibodies against the respective proteins. Endogenous proliferating-cell nuclear antigen levels were determined for loading control. From the remaining cells, ubiquitylated proteins were isolated by affinity chromatography and ubiquitylated forms of p53 were detected by Western blot analysis using a p53-specific antibody. The running positions of ubiquitylated forms of p53 are indicated by a double asterisk. The running positions of putative ubiquitylated forms of p53 detected in a whole-cell extract with a p53-specific antibody (most upper panel) are indicated by dots. Where indicated, transfected cells were treated with the proteasome inhibitor MG132 for 5 h prior to the preparation of cell extracts. (B) The effect of hHR23 overexpression on E6-mediated degradation of p53 was determined in cotransfection experiments as described in panel A for Mdm2-induced p53 degradation in the absence of an expression vector for His-tagged ubiquitin.