Fig 1: Effects of KCTD1 on ß-catenin ubiquitination and the expression of Wnt/ß-catenin downstream genes and AP-2a.(A) HeLa cells were transfected with either pCMV-Myc-ß-catenin alone or with pCMV-Myc-KCTD1 or with pCMV-Myc-TrCP or with both pCMV-Myc-KCTD1 and pCMV-Myc-TrCP. Total cell extracts were analyzed by immunoblotting using mouse monoclonal anti-Myc antibodies. ß-actin was used as the internal control. (B) HeLa cells were transfected with either pCMV-Myc-ß-catenin alone or with pCMV-Myc-KCTD1, pCMV-Myc-ubiquitin or pCMV-Myc-TrCP or in combination as indicated. 24 h after transfection, cell lysates were immunoprecipitated with rabbit polyclonal anti-ß-catenin antibodies followed by immunoblotting with mouse monoclonal antibodies against Myc-tag and ubiquitin to detect ubiquitin conjugation. (C) HeLa cells were transfected with either pCMV-Myc-ß-catenin alone or with pCMV-Myc-KCTD1 for 24 h. The whole cell extracts were valuated by Western blotting using mouse monoclonal antibodies against Myc-tag, CCND1 and AP-2a. ß-actin was used as a loading control for total lysate samples. (D) HEK293 cells were transfected with A2-Luc reporter plasmid and expression plasmids encoding KCTD1 and ß-catenin. Relative luciferase activities represent mean ±SD from at least three independent experiments after normalization to ß-galactosidase activities. **, P<0.01 compared with controls.