Cerberus protein binds to Xnr-1, BMP-4 and Xwnt-8. a, Cer–Flag secreted by Xenopus animal cap (AC) and cultured 293T cells. b, The two Cer protein products. c, HA-tagged activin, Vg1 and Xnr-1 secreted by Xenopus oocytes. d, Xnr-1 is bound specifically by Cer-S–Flag (lanes 2−4) or Cer-L–Flag (not shown). e, Cer binding inactivates Xnr-1 signalling. Animal cap explants were treated with oocyte control medium, activin (15 pM), Vg1 (0.3 nM) or Xnr-1 (1 nM) proteins, either alone (lanes 2−5) or together with 5 nM Cer-S (lanes 6−8) or Cer-L (lanes 9−11). f, Cer inhibits Xnr-1 with high affinity; 2 nM Xnr-1 was incubated with increasing concentrations of Cer-S (closed circles) or Cer-L (open circles). g, Cer-L–Flag binds BMP-4 with a KD of 0.6 nM. h, Binding of Cer-L–Flag to BMP-4 is competed for by BMP-2 but not by TGF-β-1, EGF or PDGF; Cer-S does not bind BMP-4. i, Cer-L (10 nM), but not Cer-S (20 nM), is a direct neural inducer of animal cap cells sensitized by brief dissociation–re-aggregation (but not of intact caps). j, Cer inhibits Xwnt8 but not β-catenin mRNA; induction of Siamois, a target of Wnt signalling, was assayed at stage 10+. k, Lanes 1, 2, soluble Xwnt-8–HA protein is secreted by mRNA-injected oocytes. Lanes 3−5, binding of Xwnt-8-HA (5 nM) to Cer–Flag proteins (10 nM). l, m, The Xwnt-8–HA, Xnr-1–HA and BMP binding sites in Cer-L are independent. Cer-L–Flag (2 nM) was bound to Xnr-1 (1 nM) or Xwnt-8 (2 nM) and competed with BMP-4 (10 nM), BMP-2 (27 nM) or Xnr-1 (8 nM). n, Multiple ligand-binding sites on Cerberus.