Figure 1.

Silencing of En-1 expression stimulates β-catenin transcriptional activity. (A) Si-En-1 decreases the expression of the native En-1. 293T cells were cotransfected with si-En-1 or empty pSUPER (4 μg). At the indicated times after transfection, 100 μg of cell lysates were analyzed by Western blotting using anti-En-1 or anti-β-actin antibody. (B) si-En-1 decreases the level of expression of ectopically expressed En-1. 293T cells were cotransfected with si-En-1 or scr-En-1 (2.9 μg) and En-1-HA or mEn-1-HA (0.01 μg). Seventy-two hours after transfection, 100 μg of cell lysates was analyzed by Western blotting using anti-HA or anti-β-actin antibody. (C) LiCl-activated β-catenin transcriptional activity is stimulated in the presence of si-En-1. 293T cells were cotransfected with si-En-1, scr-En-1, or empty vector (2.5 μg), TCF/Luc reporter (1 μg) and β-Gal (0.1 μg). Twenty-four hours after transfection, cells were treated with 30 mM LiCl and after 48 h, cell lysates were measured for the levels of luciferase and β-Gal activities. Data are presented as mean values and standard deviations for at least three independent experiments done in duplicate, compared with the level of luciferase activity obtained in the absence of LiCl treatment presented as 1 (top). Cell lysates (100 μg) were subjected to Western analysis using anti-β-catenin or anti-β-actin antibody (bottom).