Figure 3

F-TrCP-Ecad promotes β-catenin degradation and inhibits Wnt signaling in DLD1 cells. A. Effects of F-TrCP-Ecad on the levels of cytosolic and membrane-associated β-catenin. A DLD1 inducible cell line was generated with the tetracycline (tet)-off system; these cells expressed F-TrCP-Ecad only in the absence of Dox. Cells were grown in the presence or absence of Dox for 5 days, and subjected to subcellular fractionation. Equal amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the anti-β-catenin antibody. α-tubulin levels were determined by immunoblotting with the anti-α-tubulin antibody as an internal loading control. Quantified representation of immunoblot is shown at the bottom of each blot (relative intensity). B. F-TrCP-Ecad inhibits the transcriptional activity of β-catenin. DLD1-F-TrCP-Ecad cells were transfected with TOP-FLASH and a CMV-Renilla reporter, and cultured in the presence or absence of Dox. The luciferase activity of cells grown in the presence of Dox was arbitrarily set to 1. C. F-TrCP-Ecad induction inhibits c-MYC expression. DLD1-F-TrCP-Ecad cells were cultured in the presence or absence of Dox for four days. The expression level of c-myc and F-TrCP-Ecad were determined by immunoblotting with the anti-MYC (C-19) and the anti-FLAG antibodies (top and middle panels). Equal loadings were confirmed by immunoblotting with the anti-α-tubulin antibody (bottom panel). 'o' indicates two unknown species immuno-reactive with the anti-FLAG antibody, which migrated right below βTrCP-Ecad of the middle panel.