FIG. 3.

K345 of β-catenin is important for β-catenin/Tcf4 binding. (A) HeLa cells were cotransfected with either T41A-β-catenin, T41A-β-catenin K345R, or T41A-β-catenin K345A (5 μg) and with Tcf4-HA expression vector (2.5 μg). Proteins were extracted in lysis buffer A. Following coimmunoprecipitation (IP) with anti-β-catenin antibodies, proteins were resolved on SDS-PAGE, and Tcf4 was detected by Western blotting (WB) with anti-HA antibodies. The amounts of β-catenin in precipitates and Tcf4 in total cell lysates were shown by Western blotting with anti-β-catenin and anti-HA antibodies. (B) HeLa cells were cotransfected with the TOPFLASH reporter (0.1 μg) and T41A-β-catenin or the indicated K345 mutant (0.2 μg) or the empty vector. Luciferase activities were assayed 48 h after transfection. The basal activity of the TOPFLASH plasmid cotransfected with empty vector was arbitrarily set at 1, and other values were calculated accordingly. The data shown are the averages of three separate experiments done in duplicate. (Bottom) Expression levels of the different constructs determined by Western blotting with anti-β-catenin antibodies. (C) HeLa cells were transfected with T41A-β-catenin or the corresponding K345R or K345A mutant. Cellular extracts were prepared in buffer B, and coimmunoprecipitation assays were performed with anti-β-catenin monoclonal antibodies. Immunoprecipitates were resolved on SDS-PAGE, and blots were hybridized with either APC (left) or Axin (right panel) antibodies. Immunoprecipitated β-catenin levels were shown by Western blotting with anti-β-catenin antibodies.