Gpr183 cooperates with Arrb1 and Nedd4 to downregulate Notch signaling. (A) Internalization of Gpr183-GFP in 90% epiboly embryos (n = 6 for each treatment). White arrowheads denote the internalized Gpr183-GFP in cytoplasm. (B) Gpr183 interacts with Arrb1 in 7α-25-OHC-stimulated cells. 293T cells were transfected with Gpr183-Flag and/or Arrb1-Myc and stimulated with 7α-25-OHC for 0.5 h. Cell lysates were subjected to immunoprecipitation using anti-Myc followed by western blot analysis. (C) Expression of runx1, cmyb and hey2 in arrb1 morphants. Red arrowheads label the staining signals in AGM. (D) NICD expression in zebrafish embryos treated with 7α-25-OHC (from 1-cell stage to 36 hpf) or MG132 (from 26 to 36 hpf). Right panel shows the quantification analysis. (E) MS analysis of Notch1-interacting proteins in control or 7α-25-OHC-treated AGM cells. Mouse E10.5 AGM cells were cultured in the presence of hematopoietic cytokines for three days, and then treated with DMSO or 7α-25-OHC for 1 h. (F) Polyubiquitination of NICD-Flag (zebrafish) induced by zebrafish Nedd4a-GST (Nedd4a(Z)) or mouse Nedd4-GST (Nedd4(M)). (G) GST pull-down assay showing the interaction between Nedd4-GST and NICD-Flag. (H) runx1 expression is reduced in nedd4a morphants and increased in nedd4a-overexpressing embryos. (I) fli1a+tp1+ cells (white arrowheads) are increased in nedd4a morphants and are reduced in nedd4a-overexpressing embryos. Right panel shows the quantification result. (J) NICD expression in nedd4a morphants or nedd4a-overexpressing embryos. Right panel shows the quantification result. (K) HE in Tg(flk1:mCherry/runx1:en-GFP) embryos after nedd4a knockdown or 7α-25-OHC treatment. White arrowheads indicate HE in the AGM region at 36 hpf. Error bar represents SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.