FIGURE 6.

HBXIP up-regulates MDM2, leading to the further degradation of p53. A and B, the expression levels of HBXIP, p53, and p53 target genes such as p21 and PUMA were detected by RT-PCR in MCF-7 and LM-MCF-7 cells. C and D, the expression levels of HBXIP, p53, and p53 target genes such as p21 and PUMA were detected by Western blot analysis in MCF-7 and LM-MCF-7 cells. E, MCF-7 cells were treated with 25 mm MG-132 for 5 h, as indicated, followed by Western blot analysis. F, upper lane, MCF-7 cells were treated with 100 mg/ml cycloheximide and harvested using SDS lysis buffer at the time points indicated. The levels of p53 were analyzed by Western blot analysis, and the results were quantified using Quantity One software (Bio-Rad) as shown. Lower lane, summary of three independent experiments. G, the in vivo ubiquitination assays were performed by Western blot analysis in HEK293T cells transfected with pCMV-HBXIP plasmids. H, the in vivo ubiquitination assays were performed by Western blot analysis in MCF-7 cells transfected with pCMV-MDM2, pCMV-HBXIP plasmids, and MDM2 siRNA. I, effect of MDM2 siRNA on p53 was examined by Western blot analysis in MCF-7 cells when HBXIP was overexpressed. Each experiment was repeated at least three times.