Figure 2

F-TrCP-Ecad inhibits the transcriptional activity of β-catenin S37A. A. 293 cells were co-transfected with a β-catenin S37A expression plasmid, indicated βTrCP expression plasmids, TOP-FLASH, and a CMV-Renilla reporter. The luciferase activities were measured and normalized against the control Renilla activities (top panel). The luciferase activity of cells transfected with βTrCP was arbitrarily set to 1. The expression of βTrCP derivatives was determined by immunoblotting with the anti-FLAG antibody (bottom panel). B. F-TrCP-Ecad and F-TrCP(ΔN)-Ecad bind to β-catenin to the similar extent. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by SDS-PAGE and immunoblotted with the anti-HA antibody (top panel). The expression of FLAG-tagged βTrCP derivatives and β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-FLAG and anti-HA antibodies (middle and bottom panels).