Journal: Cancer research
Article Title: The p53 homologue ?Np63? interacts with the NF-?B pathway to modulate epithelial cell growth
Figure Lengend Snippet: ΔNp63α overexpression results in increased nuclear levels of transcriptionally active phosphorylated c-Rel in murine keratinocytes A) Elevated Δ Np63α expression specifically enhances nuclear levels of the NF-κ B subunit c-Rel. Western blots of nuclear extracts from primary murine keratinocytes harvested 21h post-adenoviral introduction of human ΔNp63α or β-gal. Cultures were maintained in medium containing 0.05mM Ca 2+ throughout or exposed to 0.12 mM Ca 2+ for the final 4h. C-Rel levels are increased in the nuclei of ΔNp63α - overexpressing keratinocytes. B) c-Rel is phosphorylated in response to elevated ΔNp63α. Western analysis of nuclear extracts from keratinocytes overexpressing ΔNp63α or β-gal cultured under control conditions (top) or in the presence of the thiol modifier N-ethylmaleimide (NEM) at concentrations noted for 21h following adenoviral introduction to block in vivo phosphorylation (bottom left). The upper species is eliminated in the presence of NEM. Phosphorylation was confirmed by western analysis of nuclear extracts derived from keratinocytes overexpressing ΔNp63α incubated in the presence or absence of Shrimp Alkaline Phosphatase (SAP) for 3h at 37°C (bottom right). A non-incubated control nuclear extract (NE) is included for visual reference. SAP treatment results in the loss of the upper phosphorylated species. C) ΔNp63α overexpression results in NF-κ B-mediated transactivation; α-domain of ΔNp63α required. NF-κB responsive reporter gene activity in keratinocytes overexpressing ΔNp63α, ΔNp63 p40 (ΔNp63 lacking α-domain) or β-gal. Samples were harvested 24h post-transfection. Results depict mean +/− s.d. of triplicate samples of a representative experiment performed thrice. D) c-Rel is required for NF-κ B-mediated transactivation following ΔNp63α overexpression. Keratinocytes were co-transfected with NF-κB reporter construct and c-Rel-targeting siRNA or control siRNA, 24h prior to adenoviral infection with ΔNp63α or β-gal. Samples were harvested 24h post adenoviral introduction. Right side: Western blot reveals depletion of c-Rel in whole cell lysates at time of harvest (NT: non targeting siRNA). Left side: Fold increase in NF-κB reporter gene activity in ΔNp63α overexpressing cultures compared to β-gal control cultures in the presence or absence of c-Rel siRNA. Triplicate wells were averaged and are presented as fold increase relative to β-gal controls that are normalized to 1.0. Experiment was performed twice with consistent results; representative experiment shown.
Article Snippet: Nuclear extracts were incubated in 1X SAP buffer +/− 10U Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, WI) for 3h at 37°C followed by inactivation at 65°C.
Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Blocking Assay, In Vivo, Derivative Assay, Incubation, Activity Assay, Transfection, Construct, Infection