Fig. 5

OTG prevents membrane targeting of Fz8 receptor and restricts Fz8 to the ER. (A,B) Immunostaining of Myc (green) in embryos (6 hpf) injected with indicated combinations of otg, fz8a-myc and fz8b-myc mRNAs. For panels A–I, nuclei are counter-stained with Hoechst 33342 (blue). (C–E) COS-7 cells were transfected for C-terminal Flag-tagged zebrafish Otg (C), human OTG (D), or human OTG with C-terminal fusion of an ER retention sequence KDEL (OTG-Flag-ER, E). Cells are immunostained for Flag (green) and an ER marker calreticulin (red). (F) Immunostaining of Myc (green) in embryos (6 hpf) injected with indicated combinations of OTG, OTG-ER and fz8-myc mRNAs. (G,H) COS-7 cells were transfected for Fz8-Myc alone (H) or with OTG (I). Cells are immunostained for Myc (green) and calreticulin (red). (I) NIH3T3 cells were transfected for Fz8-Myc and OTG-GFP. Cells are immunostained for Myc (red) and GFP (green). (J–L) Schematic representations of various deletion constructs are shown at the top of each panel. HEK293 cells were transfected for indicated combinations of OTG (OTG-flag), Otg (Otg-Flag) and Fz8-Myc. Interaction between Fz8 and OTG/Otg is examined by immunoprecipitation for OTG/Otg (IP: Flag) or Fz8 (IP: Myc) followed by immunoblotting for Fz8 (IB; Myc) or OTG (IB: Flag). Levels of proteins in total cell lysates (TCL) are also compared. Full-length blots are presented in Supplementary Fig. S19.