Figure 3. Comparison of Dkk1/2-LRP6 interaction and Wnt signaling inhibition.

Wnt reporter assay (top graph) and western blots showing endogenous Wnt pathway protein components (lower panel) in HEK293T cells treated with control medium or conditioned medium containing mouse Wnt3a, human Dkk1-GFP or human Dkk2-GFP, as indicated. 12 h after transfection with the TOPFLASH reporter plasmid construct, cells were incubated with Wnt3a or control conditioned medium for 6 h. Cells were then incubated with Dkk1/2-GFP conditioned medium as indicated for another 12 h before harvest of cell lysates for the luciferase assay. Bars show the enhancement of Wnt activity over the untreated control sample (leftmost bar set to 1); error bars represent the standard deviation from three independent experiments. In the western blots, LRP6 phosphorylation (P-LRP6) and β-catenin protein levels were assessed using specific antibodies; tubulin was used as a normalization control.