Figure 1

Wnt3a stimulates changes in cellular content of Wnt/β-catenin pathway signaling components. Panel A, formation of PE was assayed in F9 cells expressing Rfz1 and stimulated without or with purified Wnt3a for 3 days. PE formation was assayed by measuring of expression of PE-marker, cytokeratin endo A, using immunoblotting and staining with TROMA-1 antibody. The results shown are mean values ± S.E. from 3 independent experiments. A representative immunoblot is shown and the mean values of the quantification displayed in the graph. Panel B, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1 and Super8xTOPFlash (M50) or Super8xFOPFlash (M51) and then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1). The results shown are mean values ± S.E. from 5 independent experiments. Panel C, cellular abundance of Axin, Dvl2, GSK3β, p-GSK3β, PP2A C, and β-catenin was assayed in F9 cells expressing Rfz1 and stimulated with Wnt3a for 0 to 90 min. Cells were harvested and lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na₃VO₄, 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Protein expression was established by SDS-PAGE, immunoblotting, and densitometric scans of the immune complexes. A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 4 independent experiments.