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Fig. 5. Defective Notch activation in Mib1–/– embryos. (A) Notch1 intracellular domain (N1icd) generation and Hes5 expression. Lysates from E9.0 wild-type (wt) and Mib1–/– (mt) embryos were immunoblotted with rabbit anti-N1icd, mouse anti-Notch1, rabbit anti-mouse Hes5 and rabbit anti-Mib1 antibodies. The results are representative of three independent experiments. (B-D) S3 cleavage of Notch1 by {gamma}-secretase. Embryonic fibroblasts (EFs) isolated from E9.5 wild-type (wt) and Mib1–/– (mt) embryos were infected with the MSCV vector driving the expression of Myc-tagged extracellular domain-deleted Notch1 ({Delta}EN1). (B) Western blot analysis of Nicd generation. Cell lysates were immunoblotted with anti-Notch1, anti-N1icd and anti-Mib1 antibodies. HEK293A cells (HEK) were used as a positive control for infection and N1icd generation. There is intact N1icd generation in Mib1–/– EFs. (C) Nuclear transport of Nicd. Infected cells were fixed and immunostained with an anti-Myc antibody, followed by anti-mouse Alexa-488 (in green). Nuclear DNA was stained with Hoechst (in blue). N1icd are transported intra-nuclearly in Mib1–/– EFs. (D) CBF-luciferase (Luc) activation by Nicd. The 8x wild-type and mutant CBF-Luc vectors were transfected to measure the activity of N1icd in MSCV_{Delta}EN1-infected wild-type and Mib1–/– EFs. The 8x mutant CBF-Luc lacks the CBF-binding sites and was used as a control. The relative Luc activities of wild-type and Mib1–/– EFs are comparable. (E) Internalization of Delta by murine Mib1. COS7 cells were transfected with either mock GFP or HA and Myc-tagged Xenopus Delta (HA-XD-Myc) (black) or Mib1-GFP and HA-XD-Myc (green). Twenty-four hours after transfection, the cells were stained with anti-HA Ab followed by PE-conjugated anti-mouse Ab and analyzed by flow cytometry. (F) Defective Notch signaling in the Mib1–/– EFs. The wild-type (white bar) and Mib1–/– (black bar) EFs infected with MSCV vector driving the expression of Myc-tagged Xenopus Delta (XD), Myc-tagged mouse Delta1 (Dll1) or Myc-tagged mouse jagged 1 (Jag1) were co-cultured with C2C12-Notch1 cells transfected with the 8x wild-type and mutant CBF-Luc vectors. Twenty-four hours after co-culture, luciferase activity was measured. The 8x mutant CBF Luc was used as a control for Notch activation.