LRP6 levels on cell surface and in endosomes after Dkk1 and Wnt3A treatment.

<p>(A) HT1080 cells stably transduced with HA-tagged LRP6 were incubated with 25% of L cell control CM, 25% of Wnt3A CM, 0.5 µg/ml of recombinant Dkk1 protein, or 25% of Wnt3A CM plus 0.5 µg/ml of recombinant Dkk1 protein for 24 h. Cell surface LRP6 was labeled with the anti-HA antibody and detected with goat anti-mouse IgG-FITC by FACS. Each value represents the difference between total and background fluorescence intensities and is the average of triple determinations with the SE indicated by error bar. **P<0.01 compared to L cell control CM-treated cells; ^##P<0.01 compared to L cell control CM-treated cells and Wnt3A CM-treated cells. (B) HT1080 cells stably transduced with HA-tagged LRP6 were incubated with control CMs, 25% of Wnt3A CM, 25% of Dkk1 CM, or Wnt3A CM plus Dkk1 CM for 24 h. Levels of LRP6 in endosomes were examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-Rab5 antibody to verify equal loading and endosome preparation. The pixels for each band were measured, normalized and plotted. Data are mean values of four independent experiments with the SE values indicated by error bars. *P<0.05 compared to L cell control CM-treated cells; ^#P<0.05 compared to L cell control CM-treated cells and Wnt3A CM plus Dkk1C CM-treated cells.