Figure 4.

Enhancement of the interaction between β-catenin and β-TrCP by AV-65 treatment. (a) Cells were incubated with serial dilutions of AV-65 and then harvested after 6 h. Whole lysate was subjected to immunoprecipitation using an anti-β-catenin Ab, and immune complexes were captured using protein A beads. The protein A slurry (30 μl) was analyzed by immunoblot using anti-β-TrCP Ab to detect binding between β-catenin and β-TrCP. (b) IM-9 cells were transduced with lentivirus-based short hairpin RNAs targeting β-TrCP and control vector. β-TrCP levels were measured by real-time polymerase chain reaction. Data represent the means±s.d. of three independent experiments, and four replicate experiments. (c) The alterations of β-catenin expression in IM-9 cells (left panel) and β-TrCP-KD IM-9 cells (right panel) by AV-65 treatment. After 24 h incubation with serial dilutions of AV-65, cells were harvested, and were lysed in radioimmunoprecipitation assay buffer. Cell extracts (20 μg of protein) were analyzed by western blot using an anti-β-catenin Ab and an anti-β-TrCP Ab.