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Fig. 7. Disruption of E-cadherin-mediated contacts activates NF-
B transcriptional activity. (A) K-ras expression downregulates E-cadherin interaction with p65. The p65 subunit of NF-B was immunoprecipitated from total-cell extracts prepared from IEC or IEC K-ras-transfected cells. Immunocomplexes were analysed by western blot with mAbs against E-cadherin, and β-, 
- and p120-catenin. The results given correspond to a representative experiment out of the three performed. (B) E-cadherin siRNA affects the association of p65 to junctional-complex components. IEC cells were transfected with an siRNA specific to E-cadherin or with an irrelevant siRNA as control. Total extracts were prepared, the p65 subunit of NF-B was immunoprecipitated and immunocomplexes were analysed by western blot with mAbs against E-cadherin, and β-, - and p120-catenin. The results given correspond to a representative experiment out of the three performed. (C) E-cadherin siRNA increases p65 nuclear levels. Presence of p65 in the nucleus was determined by western blot analysis of cell fractions prepared from cells transfected with E-cadherin siRNAs or irrelevant siRNAs (Irr), as indicated above. Lamin B1 and TBP or pyruvate kinase were used as nuclear and cytosolic markers, respectively. (D,E) E-cadherin siRNA increases NF-B transcriptional activity. IEC cells were transfected with irrelevant or E-cadherin siRNAs and NF3 reporter plasmid. Luciferase activity was determined after 48 hours and is shown as the average ± s.d. of three experiments performed in triplicate. In E, cells were deprived of serum for 16 hours and incubated with TNF (20 ng/ml) for 6 hours before analysis of NF3 promoter activity.
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