Fig. 6. SFRP1 activities do not modify ß-catenin transcriptional activity. (A) Immunoblot analysis of cytosolic ß-catenin levels in MDCK/control or MDCK/SFRP1 treated retina cells harvested after three and five hours of culture. Cytosolic fractions were prepared and immunoblotted with antibodies against ß-catenin and -tubulin (load control). Normalized density values (NDV) expressed in arbitrary units indicate no differences among the fractions. (B) Luciferase assay showing that SFRP1 does not modulate the endogenous level of ß-catenin/TCF-dependent transcription in E5 chick dissociated retinal cells. Cells were co-transfected with the reporter plasmid containing the Lef-1 responsive element (C4) or its mutated version (MUT), the control plasmid pRLTK and the effector plasmids in each case. Either 500 ng or 1 µg of Sfrp1 were transfected alone or in combination with 500 ng of Wnt8. 500 ng of Wnt8 or ß-catenin activate the reporter gene 240 and 130 fold, respectively. The two concentrations of Sfrp1 decreased Wnt8 activity 4 and 10 fold, respectively. 1 µg of dnGSK3ß did not activate the reporter. Luciferase activity is expressed in a logaritmic scale.