Figure 6. ATDC stabilizes β-catenin by interacting with disheveled-2 and the β-catenin destruction complex.
(A) Pulse-chase assays in Panc1 cells (with or without ATDC silencing) were performed to determine β-catenin stability. (B) β-catenin remaining in (A) was quantitated by densitometry at hours 0, 3 and 6 and normalized relative to the 0 hour time point. Results are the mean ± SE of three independent experiments (*p<0.05 vs control shRNA cells at 6 hours). (C, D) Cell lysates from HEK 293 cells (C) transfected with empty vector or Flag-ATDC, and BxPC3 cells (D) were subjected to immunoprecipitation (IP) with Axin, GSK3β or β-catenin antibodies. Immunocomplexes were resolved by SDS-PAGE and subjected to western analysis with anti-Flag antibody for HEK293 cells (C) and ATDC antibody for BxPC3 cells (D). Blotting with an anti-β-actin antibody revealed equal loading. (E–G) Lysates of HEK 293 cells (E) transfected with empty vector or ATDC expression vector, and control shRNA- or ATDC shRNA1 or 2- expressing Panc1 (F) and BxPC3 (G) cells were subjected to Western blotting with an anti-Dvl-2 antibody. The upper arrow indicates the phosphorylated form and the lower arrow indicates the non-phosphorylated form of Dvl-2. Experiments were performed twice with similar results. (H) Western blotting of 5 samples each of pancreatic adenocarcinoma and normal pancreas. Dvl-1, Dvl-2, Dvl-3 and ATDC expression in pancreatic tissue samples was measured. The experiments were repeated twice with similar results. (I, J) Cell lysates from HEK 293 cells (I) transfected with empty vector or Flag-ATDC and BxPC3 cells (J) were subjected to immunoprecipitation (IP) with Dvl2 or β-catenin antibodies. Immunocomplexes were resolved by SDS-PAGE and subjected to western analysis with an anti-Flag antibody for HEK293 cells (I) and ATDC antibody for BxPC3 cells (J). Blotting with an anti-β-actin antibody showed equal loading. (K) Pulse-chase assays were performed in Panc1 cells (with or without ATDC silencing) to determine Dvl-2 stability. (L) Dvl-2 remaining in (K) was quantitated by densitometry at hours 0 and 6 and normalized relative to the 0 hour time point. Results are the mean ± SE of three independent experiments (*p<0.05 vs control shRNA cells at 6 hours).