FIGURE 2.

HBP1 promotes p21 transcription by enhancing p53 protein stability. A, HBP1 prolongs the half-life of p53. A549 cells were stably transfected with PITA, PITA-HA-HBP1 or pLL3.7, and pLL3.7-shHBP1 through lentiviral infection. Cells were incubated with the protein translation inhibitor cycloheximide (CHX) for 0, 20, 40, 60, or 80 min before harvest. P53 and Mdm2 protein levels were detected by Western blotting. GAPDH was used as the loading control for this turnover experiment (top panel). Densitometry is plotted for the average ± S.D. of three independent experiments (bottom panel). B, HBP1 does not elevate p53 levels in the presence of MG132. H1299 cells were cotransfected with p53 and GFP with or without HBP1. 42 h after transfection, cells were incubated with (third and fourth lanes) or without (first and second lanes) MG132 for another 6 h. p53 protein was detected by Western blotting. Level of GFP is shown as equal transfection efficiency. C, overexpression of HBP1 enhances the binding of p53 to the p21 promoter. H1299 cells were transfected with HA-tagged p53 and pcDNA3, pcDNA3-HA-HBP1, pcDNA3.1-His-EZH2, or both pcDNA3-HA-HBP1 and pcDNA3.1-His-EZH2. 24 h after transfection, cells were lysed for ChIP assay. The lysates were incubated with anti-p53 antibody or control IgG. The precipitated DNA fragments were amplified with specific oligonucleotides for the p21 promoter by real-time PCR. Results are representative of three independent experiments, and values are the mean ± S.E. *, p < 0.05.