Biochemical properties of sDll4. (A) Notch-Fc fusion protein was coated directly on enzyme-linked immunosorbent assay (ELISA) plates. sDll4-AP was allowed to bind Notch-Fc, and the bound Dll4 was quantitated by the addition of AP substrate. sDll4-AP bound efficiently to Notch1 and not Notch3 (left panel). Binding of sDll-4Fc and sDll4-His to Notch1 was examined. (B) HUVECs were transfected with expression vectors for sDll4-Fc, sDll4-His, or vector alone. Notch-responsive Hes-2 gene expression was not induced by sDll4 proteins. (C) Notch activation measured by the induction in Hes-1, Hey-1, and Hes-2 when HUVECs were cocultivated with ChoK expressing Dll4-FL (full length). Addition of recombinant sDll4-Fc and sDll4-His reduced the induction of Notch responsive genes. Two independent experiments produced similar results.