Notch1 lacking the ligand binding domain is expressed on the cell surface but does not signal. (A) Growth curves of ES cells isolated from E3.5 Notch1+/+, Notch1+/lbdand Notch1lbd/lbdblastocysts. Bars represent mean ± SD. (B) Western blot analysis of ES cell lysates (50 μg protein). Full length Notch1 was detected by antibody 8G10 and activated Notch1 was detected by antibody Val1744. Blots were stripped and reprobed using anti-β-Tubulin III. Data are representative of 3 experiments. (C) Flow cytometry of cell surface Notch1 in Notch1lbd/lbdand Notch1+/+ ES cells using anti-Notch1 ECD antibody 8G10 followed by Alexa-488 conjugated anti-hamster IgG. Grey profiles are secondary antibody alone. Profiles are representative of two experiments. (D) Notch ligand binding. ES cells were incubated with soluble Delta1-Fc followed by PE-conjugated anti-human IgG and analyzed by flow cytometry. Notch1in32/in32 null ES cells were line 290-2. EDTA in the binding buffer inhibited binding to all Notch receptors. Bars represent mean ± SEM; n = 5 for Notch1+/+ and Notch1lbd/lbd; n = 3 for Notch1in32/in32 ES cells. (E) ES cells were assayed for Notch signaling after transfection of the Notch TP-1 reporter construct by co-culturing with L cells expressing Delta1 or Jagged1 compared to control L cells. Bars represent fold-activation ± SEM for Notch1+/+ (white), Notch1lbd/lbdand Notch1in32/in32 (gray) (n = 4; ** P < 0.01, *** P < 0.001).