Fig. 6. Interactions between Mib1 and all known Notch ligands. (A) Co-immunoprecipitation (Co-IP) of murine Mib1 with murine Notch ligands (Dll1, Dll3, Dll4, Jag1 and Jag2). HA-tagged Mib1 (HA-Mib1) or control vectors were co-expressed with Myc-tagged Notch ligands in HEK293A cells. The top panels show IP of Notch ligands by HA-Mib1, and the middle and bottom panels show the expression of HA-Mib1 and Notch ligand-Myc, respectively, in total cell lysates. (B) Subcellular localization of Mib1 (in green) and Notch ligands (Dll1, Jag1 and XD; in red). Mib1-GFP and/or Myc-tagged Notch ligand constructs were co-expressed in COS7 cells. Myc epitopes were detected with an anti-Myc antibody followed by a TRITC-labeled antibody. Nuclear DNA was stained with Hoechst (in blue). (a-c) Expression of Mib1-GFP (a), Dll1-Myc (b) and Jag1-Myc (c). (d-f) Co-transfection of Mib1-GFP with either Dll1-Myc (d), Jag1-Myc (e) or XD-Myc (f). Overlapping expression is yellow. (C) Transverse sections of E9.0 wild-type (wt) and Mib1^–/– (mt) embryos stained with anti-Dll1 antibody. Dll1 is localized in the cytoplasm in wild-type neural tube, and accumulates in the plasma membrane in Mib1^–/– neural tube (in red). Nuclear DNA was stained with Hoechst (in blue). The overall images are shown in insets. (D) Notch signal transduction. The pan-Notch phenotype in Mib1^–/– embryos and the molecular interactions between Mib1 and multiple Notch ligands suggest a new core-Notch component that regulates the endocytosis of Dll and Jag ligands. The endocytosis of the Notch ligands by Mib1 stimulates the S2 and S3 cleavages of Notch receptors and the released Nicd translocates to the nucleus to express the Notch target genes, such as Hes5, Hey1 and Heyl.