Fig. 7. Synergistic activation of Tcf-4 by β-catenin and PIASy. (A) Activation of Tcf-4 by β-catenin and PIASy. The indicated amounts of pCMV5-Flag/PIASy, pEF-BOS/hTcf-4E (0.1 µg) and TOP-fos-Luc (0.5 µg) were transfected into 293 cells with or without pUC/EF-1α/β-cateninSA (10 ng) (left panel). The indicated amounts of pUC/EF-1α/β-cateninSA, pEF-BOS/hTcf-4E (0.1 µg) and TOP-fos-Luc (0.5 µg) were transfected into 293 cells with or without pCMV5-Flag/PIASy (0.1 µg) (right panel). The luciferase activity was measured and expressed as the fold increase compared with the level observed in cells transfected with TOP-fos-Luc and pEF-BOS/hTcf-4E. Left panel: Flag-PIASy alone (open circles); β-cateninSA and Flag-PIASy (filled circles). Right panel: β-cateninSA alone (open squares); Flag-PIASy and β-cateninSA (filled squares). The lysates (20 µg of protein) of 293 cells expressing the indicated proteins were probed with the anti-HA, anti-Flag and anti-Tcf-4 antibodies (lower panel). (B) pUC/EF-1α/β-cateninSA (50 ng) (lanes 2, 5, 6, 8, 11 and 12), pCMV5-Flag/PIASy (0.5 µg) (lanes 3, 5, 9 and 11) and pCMV5-Flag/PIASyCA (1 µg) (lanes 4, 6, 10 and 12) were transfected into 293 cells with pEF-BOS/hTcf-4E (lanes 1–12) and TOP-fos-Luc (lanes 1–6) or FOP-fos-Luc (lanes 7–12). The lysates (20 µg of protein) of 293 cells expressing the indicated proteins were probed with the anti-HA and anti-Flag antibodies (lower panel). (C) Activation of Lef-1 by β-catenin and PIASy. The assay conditions were the same as those in (B) except that pCMV5-T7/Lef-1 (0.1 µg) was used instead of pEF-BOS/hTcf-4E (0.1 µg). (D) Effects of PIASy on Tcf-4-(Δ1–53). pUC/EF-1α/β-cateninSA (10 ng) (lanes 2, 4, 6 and 8) and/or pCMV5-Flag/PIASy (0.5 µg) (lanes 3, 4, 7 and 8) were transfected with TOP-fos-Luc and pEF-BOS/hTcf-4E (0.1 µg) (lanes 1–4) or pEF-BOS/hTcf-4E-(Δ1–53) (0.5 µg) (lanes 5–8). (E) Synergistic activation of endogenous Tcf family proteins by β-catenin and PIASy. The indicated amounts of pCMV5-Flag/PIASy and TOP-fos-Luc (0.5 µg) were transfected with or without pUC/EF-1α/β-cateninSA (10 ng). Flag-PIASy alone (open circles); β-cateninSA and Flag-PIASy (filled circles). (F) Effects of PIASy and β-catenin on the Tcf activity for natural Tcf-responsive promoters. pUC/EF-1α/β-cateninSA (0.5 µg) (lanes 2, 5, 6, 8, 11 and 12), pCMV5-Flag/PIASy (0.5 µg) (lanes 3, 5, 9 and 11) and pCMV5-Flag/PIASyCA (1 µg) (lanes 4, 6, 10 and 12) were transfected with cyclin D1(-163)-Luc (0.5 µg) (lanes 1–6) or Axin2-Luc (0.5 µg) (lanes 7–12). (G) Effect of PIASy on Wnt-3a- or Dvl-1-dependent Tcf activity. pPGK/Wnt-3a (0.5 µg) (lanes 2, 5 and 6) or pCGN/Dvl-1 (0.5 µg) (lanes 8, 11 and 12) was transfected with TOP-fos-Luc and pCMV5-Flag/PIASy (0.5 µg) (lanes 3, 5, 9 and 11) or pCMV5-Flag/PIASyCA (1 µg) (lanes 4, 6, 10 and 12). All the experiments were performed at least three times, and the results shown are means ± SE.