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Fig. 6
OTG regulates glycosylation of Fz8 receptor. (A–D) Immunostaining of Myc (green) in embryos (10 hpf) injected with fz8-myc mRNA alone (A), with OTG mRNA (C), with otg MO (D) or treated with an N-glycosylation inhibitor tunicamycin (B). For panels A–D and G–I, nuclei are counter-stained with Hoechst 33342 (blue). (E) HEK293 cells were transfected for Fz8-Myc and increasing amounts of OTG-Flag. Overexpressed Fz8-Myc (anti-Myc) and OTG-Flag (anti-Flag) proteins are analyzed by immunoblotting. (F) HEK293 cells were transfected for Fz8-Myc and then treated with tunicamycin, or their lysates were incubated with endoglycosidase H (Endo H) or O-glycosidase (O-Gly). Overexpressed Fz8-Myc protein (anti-Myc) is analyzed by immunoblotting. Full-length blots are presented in Supplementary Fig. S19. (G–I) HEK-293 cells were transfected with Fz8-Myc and then treated with vehicle DMSO alone (G) or with tunicamycin [0.1 μg/ml (H) and 1 μg/ml (I)]. Cells are immunostained for Myc (green).