FIG. 6.

Pin1 antagonizes the inhibition of Tcf4 signaling by the DHT-liganded AR. (A to D) 293T cells were transfected with pRL-CMV (1 or 2.5 ng), pTopflash (20 ng), pCIneo-AR, and β-catenin expression vectors as indicated (+, used; −, not used). Total DNA was normalized using empty pcDNA3 in panel A, pBluescript or empty pcDNA3 in panel B, and a mixture of empty pcDNA3 and pBluescript in panel C (with the amount of pcDNA3 adjusted to be equimolar in each sample). Results are given in relative light units (RLU). (E to G) 293T cells were transfected with 3 μg of β-catenin and Tcf4 plasmids in every case, 3 μg of AR as indicated, and 3 or 9 μg of Pin1 vector as indicated. pcDNA3.1 vector was used to equalize the total plasmid amount. Cell lysates were precleared and then immunoprecipitated (IP) with control nonimmune mouse serum (E) or mouse anti-Tcf4 antibody (F), followed by immunoblotting for β-catenin. The position of β-catenin is indicated with an arrow, while the lower band (*) is an immunoglobulin (IgG) dimer present in the anti-Tcf4 antibody preparation that is recognized by the secondary anti-mouse antibody alone (not shown). (G) Inputs (1%) for the indicated proteins.