Figure 5.

14-3-3 τ and σ stabilize p53. (A) H1299 cells expressing inducible p53 were transfected with 14-3-3 isoforms, followed by induction of p53 for 24 h (in tet-free medium). p53 induction was stopped by placing the cells in tet-containing medium and cells were treated with 0.5 µM CPT or DMSO (control) for an additional 24 h. Cell extracts were prepared and resolved by SDS–PAGE followed by immunoblotting for p53 using PAb1801 antibody. γ and ε had no effect on the p53 levels while τ and σ increased p53 levels. (B) Dose–response luciferase assay for 14-3-3 τ and σ is shown. No stimulation of luciferase activity was observed when p53 is not induced. (C) H1299 cells were transfected with increasing amounts of 14-3-3 τ and σ. Twenty-four hours post-transfection, cells were lysed and immunoblotted with anti-τ, anti-σ and anti-p53. A notable increase in p53 level was observed. (D) Dose–response luciferase assay for 14-3-3 γ and ε is shown. No stimulation of luciferase activity was observed when p53 is not induced. (E) Immunoblotting of cell lysates from H1299 cells transfected with increasing amounts of 14-3-3 γ and ε showed no pronounced effect on the p53 levels.