FIGURE 4.

Signaling of ligand truncations. A, non-reducing SDS-PAGE gel of purified Dll1 proteins. B, non-reducing SDS-PAGE gel of purified Dll4 proteins. Multiple bands are present as a result of N-linked glycosylation, which was not observed in Dll1. C, signaling induced by Dll1 truncations. Purified Dll1 proteins were plated at an equivalent molar concentration and used to activate N1gal4 U2OS stable cells. Resulting luciferase activity is shown on the y axis, normalized to activation with plated human IgG. C-terminal truncation up to EGF3 had little effect on signaling, whereas removal of the N-terminal MNNL domain (ΔMN) abolished activity. D, signaling induced by Dll4 truncations, performed as in C. C-terminal truncations up to EGF3 also maintained full signaling capacity, whereas removal of EGF3 abolished signaling. E and F, concentration dependence of ligand-induced signaling. Dll1(1–5) and Dll4(1–5) were plated at varying concentrations and incubated with U2OS cells transfected with either full-length N1gal4 or N1gal4(6–15). The resulting luciferase activity is shown as a function of concentration, fitted with a one-site exponential binding model. Calculated EC₅₀ values for N1gal4 were 227 ± 175 nm (Dll1) and 47 ± 22 nm (Dll4); for N1gal4(6–15), 281 ± 82 nm (Dll1) and 47 ± 33 nm (Dll4).