FIG. 7.

β-TrCP mediates Smad3-induced Cdc25A ubiquitination. (A) Smad3 knockdown diminishes Cdc25A interaction with β-TrCP. U2OS cells were transfected with Smad3 siRNA or NS dsRNA, followed by transfection with Flag-tagged β-TrCP2 and/or Cdc25A. Cells were treated with 2 μM MG132 at 16 to 24 h posttransfection and subjected to immunoprecipitation (IP) followed by immunoblotting (WB), as indicated. Direct WB data are shown in lower panels. Asterisks, immunoglobulin heavy chain. (B) siRNA-mediated down-regulation of β-TrCP protects Cdc25A from Smad3-mediated degradation. U2OS cells were transfected twice with NS, Smad3, β-TrCP2, or Cdh1 siRNA. At 24 h posttransfection, cells were further transfected with either Cdc25A alone or Cdc25A and Smad3, and 24 h later, protein levels were determined by immunoblotting.