FIGURE 2.

Signaling of N-terminal Notch truncations. A and B, ligand-induced activation of N-terminal N1gal4 truncations. U2OS cells were transiently transfected with receptor variants, co-cultured with ligand-expressing or control cells, and the resulting activation was measured via luciferase activity. A, Notch N-terminal truncations activated with OP9-Dll1 cells. Gradual loss of activity can be seen with removal of EGF repeats 6 and 7, and complete loss of activity with removal of EGF8. B, Notch N-terminal truncations activated with MS5-Dll4 cells. Activity equivalent to full-length N1gal4 is observed upon deletion of EGF repeats 6 and 7, but deletion of EGF8 abolishes signaling. All results are shown normalized to co-culture with non-ligand-bearing OP9 or MS5 cells, and treatment with the γ-secretase inhibitor compound E (GSI, 1 μm) inhibits activation in all receptor constructs tested. C, surface expression of truncated receptors. Surface expression was detected via flow cytometry, using an antibody to the Notch1 NRR (WC-75) and a FITC-conjugated secondary antibody. Percentage of Notch1-positive cells is indicated on the y axis.