Fig. 1. Identification of A23187 as a small-molecule inhibitor of Wnt/ß-catenin signaling. (A) Screening of compounds that inhibit Wnt/ß-catenin signaling. (B) Dose-dependent inhibition of CRT induction plotted as increasing concentrations of A23187. HEK293 reporter and control cells were incubated with A23187 (0.625, 1.25, 2.5 and 5 µM) for 15 hours in the presence or absence of Wnt3a-CM, and luciferase activity was determined. The results shown are the average of three experiments; the bars indicate standard deviations. (C) Cytosolic proteins were prepared from HEK293 reporter cells treated with either vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM and were then subjected to western blotting with anti-ß-catenin antibody. (D) Cytosolic proteins prepared from HEK293 reporter cells were incubated with vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM, exposed to MG-132 (20 µM) for 8 hours, and then subjected to western blotting with anti-ß-catenin antibody. In C and D, the blots were reprobed with anti-actin antibody as a loading control.