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. 2005 Apr;25(8):3338–3347. doi: 10.1128/MCB.25.8.3338-3347.2005

FIG. 6.

FIG. 6.

Association of β-TrCP with Cdc25A and Smad3. (A) Cdc25A physically associates with the F-box proteins β-TrCP1 and β-TrCP2 in cotransfected U2OS cells. Cells were transfected with Cdc25A and Flag-tagged β-TrCP1 or β-TrCP2 and treated with 2 μM MG132 at 16 to 24 h posttransfection. Lysates were immunoprecipitated (IP) with an anti-Flag monoclonal antibody, followed by immunoblotting (WB) for Cdc25A. Asterisk, immunoglobulin heavy chain. The lower panel shows direct WB. (B) TGF-β up-regulates Cdc25A association with β-TrCP. Cells were transfected as described for panel A, followed by treatment with 10 ng of TGF-β/ml1 for 24 h. MG132 (1 μM) was added for the last 15 h of incubation. Asterisk, immunoglobulin heavy chain. The lower panel shows direct WB. (C) Smad3 physically associates with β-TrCP. Cells were transfected with the indicated plasmids, followed by treatment with 2 μM MG132 at 16 to 24 h posttransfection. Asterisks, immunoglobulin heavy chain. The lower three panels show direct WB. (D) Smad3 interacts with cullin-1 (Cul1). Cells were transfected with Cdc25A, Smad3, and Cul1-V5 as indicated, followed by the treatment with 2 μM MG132 at 16 to 24 h posttransfection. Lysates were immunoprecipitated with anti-Smad3 antibody followed by immunoblotting with V5 antibody for cullin-1. Asterisk, immunoglobulin heavy chain.