Figure 1.

CPE affects the Wnt signaling pathway. (a) HEK293T cells were co-transfected with the pTOPFLASH/pFOPFLASH reporters and the β-gal plasmid, along with GFP-β-catenin and F-CPE-GFP or ΔN-CPE-GFP as indicated (0.5 μg/ml). Forty-eight hours later the cells were harvested and luciferase levels were determined (upper panels). The same samples were subjected to western blot analysis using an anti-GFP antibody. β-Galactosidase activity was used to normalize transfection efficiency. Tubulin served as a loading control. (b) As in a, showing dose-dependent activity of the F-CPE-GFP construct on β-catenin and Wnt signaling. (c) HEK293T cells were co-transfected with the pTOPFLASH/pFOPFLASH reporters, the β-gal plasmid and F-CPE-GFP, Fz1 + Wnt3a or Dvl as indicated. Forty-eight hours later the cells were harvested and luciferase levels were determined (upper panel). The same samples were subjected to western blot analysis. F-CPE-GFP protein was detected using an anti-GFP antibody, and endogenous active β-catenin was detected using an anti-active β-catenin antibody. Tubulin served as a loading control. (d) Using transient transfection of specific CPE siRNA-encoding oligos, the expression of the endogenous F-CPE protein in PC-12 cells was decreased. Twenty-four hours post siRNA transfection the cells were transfected with pTOPFLASH/pFOPFLASH and β-gal plasmids for luciferase detection. At 48 h post transfection, the cells were harvested and subjected to luciferase assay according to the manufacturer's instructions followed by western blot analysis as described above. The endogenous CPE, c-myc and SOX-9 were detected using specific antibodies. Endogenous active β-catenin was detected using an anti-active β-catenin antibody. Tubulin served as a loading control.