Figure 4: The interaction between Cdo and Lrp6 was mediated by the second immunoglobulin of Cdo and LDLR domains of Lrp6 in the ectodomain.

(a) Lysates of 293T cells co-transfected with Lrp6 and expression vectors for either a control or the fusion protein of Cdo’s intracellular region to GST (GST-CdoIN) were subjected to a pull-down assay with glutathione–agarose beads and analysed by immunoblotting. (b) Protein A-agarose pull-down assays of 293T cells co-transfected with expression vectors for Lrp6 and various Fc fusion proteins containing the ectodomain of Cdo, Boc or Neogenin. (c) Protein A-agarose pull-down assays of 293T cells transfected with expression vectors for a Lrp6 ectodomain–Fc fusion protein Lrp6 (Fc) and various Cdo deletion mutants. (d) Protein A–agarose pull-down assays of 293T cells co-transfected with Lrp6 (Fc) and the full length or Ig2 deletion mutant of Boc. (e) Co-immunoprecipitation of 293T cell lysates transfected with expression vectors for Cdo and three different myc-tagged Lrp6 extracellular domains; EGF-like domains 1–2 (E1–2), E3–4 or LDLR domain (L1–3) with a myc antibody. All experiments were repeated at least three times with similar results.