Figure 2.

Alteration of β-catenin expression in myeloma cells by AV-65 treatment. (a) Cells were incubated with serial dilutions of AV-65 and harvested after 6, 12 and 18 h. Nuclear and cytoplasmic protein fractions were obtained and analyzed by western blot using an anti-β-catenin Ab. Results are representative of three independent experiments. (b) Ubiquitination of β-catenin by AV-65 treatment. Cells were incubated with the indicated concentration of AV-65 (1 × IC₅₀), and then harvested after 12 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-mono- and anti-poly-ubiquitinyl conjugates to detect ubiquitination of β-catenin (left panel). The bands of about 60 and 30 kDa detected in the membrane using anti-β-catenin Ab are nonspecific (right panel).