FIG. 7.

Association of C1 and C2 with LRP6. (A) Cell culture media with Dkk-GFP proteins were incubated for 1 h with HEK293T cells transfected either with LRP6 (LRP6) or a control vector (−) as indicated. GFP fluorescence reflects Dkk-GFP binding to cells. Bar, 65 μm. (B) LRP6-transfected HEK293T cells were preincubated for 30 min with the culture media containing C1, C2, or N1, followed by additional 30-min incubation with Dkk-GFP-containing media as indicated. Bar, 65 μm. (C) Biochemical association of C1 and C2 with LRP6. Media from HEK293T cells, expressing the extracellular domain of LRP6 fused to the IgG heavy chain (LRP6) or control secreted IgG heavy chain (IgG) were mixed in equal volumes with Dkk-containing media as indicated and incubated with protein A-Sepharose beads (ProtA) to bind IgG or LRP6. The presence of Dkks in ProtA pellets and original supernatants (Supn) was assessed with anti-Flag or anti-HA antibodies, and LRP6 and IgG were detected with anti-human IgG (Fc) (Anti-hIgG) antibody.