Binding of Wnt3a mutants to Fz8-CRD-Fc. (A) Binding efficiency of mutants from sites 1, 2 and 3, and double mutants were analyzed by precipitation from a conditioned medium with recombinant Fz8-CRD-Fc or Fc alone pre-bound to Protein G agarose beads. Bound protein fractions were analyzed by immunoblotting using anti-Wnt3a antibody. (B) Pull-down assay of wild-types Wnt3a and site 1 mutants S209A and G210R with Blue Sepharose beads. BS, Blue Sepharose;s IgG, immunoglobulin G; IP, immunoprecipitation; CM, Conditioned Medium.