FIG. 5.

14-3-3σ enhanced p53 transcriptional activity. (A) 14-3-3σ activated the p53 luciferase reporter gene. The BDS2-3X-luc reporter containing a p53-responsive element was transfected with the indicated p53- or 14-3-3σ-expressing vectors into R1B/L17 cells. Relative luciferase activity is shown. (B) 14-3-3σ potentiated p53 transcriptional activity in a dose-dependent manner. The BDS2-3X-luc reporter was transfected with increasing amounts of the 14-3-3σ expression vector into p53-null MEF cells. The BDS2-mutant-luc reporter with a mutated p53-responsive element was used as a negative control. Relative luciferase activity is shown. (C) Oligomerization assay. H1299 cells were transfected with the indicated plasmids. Total cell extracts were incubated with 0.01% glutaraldehyde to determine the levels of the oligomerization of p53. p53 oligomers were immunoblotted (I.B.) with anti-p53 antibodies. (D) 14-3-3σ increased the interaction between p53 molecules. H1299 cells were transfected with pCMV-His-tagged-p53 and pCMV-GFP-p53 in the presence or absence of 14-3-3σ. His-tagged p53 was bound with His-Bind beads, and His-tagged p53-associated GFP-p53 was observed with the anti-GFP antibody. (E) Transcriptional activation of p53 target gene p21. R1B/L17 cells were infected (inf) with Ad-14-3-3σ (σ) or Ad-β-gal (C) or not infected (−). After 48 h, total RNAs were prepared. Northern blot analysis was performed on total RNAs to examine the expression of the p21 gene. Signals for GAPDH are shown to indicate the integrity and quantity of the RNA.