Ryk processing and domain localization. (A) Schematic of three different Ryk expression constructs. The positions of C155 and C188 are noted. SP, signal peptide; WIF, Wnt-inhibitory factor domain; TM, TM domain; TyrK, tyrosine kinase domain. (B) Western blot analysis of Flag-Ryk-myc with increasing amounts of Wnt5b reveals Ryk cleavage with ICD and turnover. Tagged Ryk RNA was injected at 20 pg; Wnt5b RNA was injected at 5 (+) and 10 pg (++). (C) Western blot analysis of Flag-Ryk-myc under nonreducing conditions showing differential accumulation of Ryk full-length (FL) and ECD forms with wild-type (WT) and Ryk C155A ECD expression constructs. ryk RNA was injected at 20 pg; ryk C155A RNA was injected at 20 (+) and 50 pg (++). (D) Coimmunoprecipitation analysis showing that Wnt5b-myc interacts with Ryk ECD-TM-EGFP and not with Ryk C155A mutant. Wnt5b-myc was injected at 10 pg; wild-type and C155A ECD-TM-EGFP were injected at 50 pg. IB, immunoblot; IP, immunoprecipitation. (E–G) In donor embryos, Flag tag detects ECD (E), and C-terminal monomeric EGFP detects ICD (F) at 80–90% epiboly; merged images are shown (G). (H–J) After transplantation into a Wnt5b-expressing host, both ECD (H) and the ICD (I) show internalization; merged images are shown in J and coarse colocalization (asterisk). Note increased protrusive activity of the transplanted cell (J, arrowhead). Bar, 20 µm.