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Fig. 9. Physical and functional interaction of Xgly4 with Wnt11. (A) Xgly4 binds and co-precipitates Wnt11, Wnt5a or Wnt8. HA-tagged Wnts expressed in HEK293T cells were efficiently co-precipitated with Flag-tagged full-length Xgly4 as well as a Flag-tagged extracellular domain of Fz7. (B) Xgly4 interacts efficiently with the signaling receptor Fz7, but barely with ActRIB (ALK4) or with ActRIIA. (Top) Pull-down of each GST-fused receptor expressed in 293T cells with glutathione-Sepharose, followed by western blotting with anti-Flag antibody. (Middle) Expression levels of Xgly4 revealed with anti-Flag antibody. (Bottom) Expression levels of receptors revealed with anti-GST antibody. (C) The cysteine-rich domain (CRD) of Xgly4 protein functionally interacts with Wnt11 signaling. (Top) The construction of wild-type and mutant Xgly4 proteins. Line represents internal deletion, black box designates epitope flag-tag sequence, asterisks indicate the putative glycosylation sites and C represents cysteine residues. (Bottom) Activin-induced elongation (bottom left) was inhibited by co-injection with wild-type Xgly4 (500 pg; top middle), Xgly4{Delta}GAG (500 pg; bottom middle), or XGly4{Delta}C (500 pg; top right), but not with XGly4{Delta}CRD (500 pg; bottom right) mRNA.