Fig. 6. NF-B associates with E-cadherin and other components of the junctional complex. (A) NF-B co-immunoprecipitates with E-cadherin and β-catenin. The p65 subunit of NF-B was immunoprecipitated from whole-cell extracts of SW-480 cells stably transfected with Snail1-HA and E-cadherin. The associated proteins were analysed with specific mAbs against E-cadherin, and β-, - and p120-catenin. (B)NF-B associates with E-cadherin and E-cadherin-associated proteins. Pull-down assays were performed by incubating 5 pmol of the different GST-fused proteins with 500 µg of cell extracts from confluent SW-480 cells prepared in RIPA buffer. Protein complexes were affinity-purified with glutathione-Sepharose and analysed by western blotting with anti-p65 mAb. Blots were re-analysed with anti-GST antibodies to ensure equal loading of samples. 3% of the total-cell extracts used for the assay was loaded in the input lane. When indicated, 25 pmols of cytoE-cadherin recombinant protein were added to the binding assays. (C) Recombinant purified p65 [1 (+) or 2 (++) pmol] was incubated with 5 pmol of either cytosolic fragment of E-cadherin fused to GST (GST–cytoE-cad) or GST as a control in the absence or presence of 500 µg of SW-480 cell extracts. Protein complexes were affinity-purified with glutathione-Sepharose and analysed by western blot as above. Known amounts of extract (3 µg) and recombinant p65 (0.05 pmol, 3 ng) were included in the same blot as controls (Input). (D) NF-B interacts only with E-cadherin-associated β-catenin. Extracts, prepared as in A, from IEC-18 cells expressing E-cadherin were immunoprecipitated with a mAb specific to E-cadherin in two successive rounds. The presence of p65, E-cadherin and β-catenin was analysed before and after E-cadherin immunodepletion by western blot (input). Extracts before or after immunodepletion were immunoprecipitated with mAbs against E-cadherin, β-catenin or an irrelevant IgG as control and presence of E-cadherin, β-catenin or p65 in the immunocomplex was determined by western blot. (E) IB prevents association of NF-B with E-cadherin. NIH3T3 fibroblasts were transfected with 7.5 µg of pcDNA3 control, pcDNA3–E-cadherin or pCdna3-IB S32,S36 mutant (a kind gift from A. Bigas, IDIBELL, Barcelona, Spain). After 48 hours, cell extracts were prepared and the p65 subunit of NF-B was immunoprecipitated. The presence of E-cadherin, catenins and IB in the complexes was analysed by western blot with specific mAbs. The results shown correspond to a representative experiment out of the three performed.