Fig. 4. Nti function is essential for Notch receptor to bind Delta ligand. (A) Co-expression of Nti and Fng with Notch synergistically increases the binding to Delta-AP. (Upper panel) Delta-AP binding to transfected S2 cells as measured by an enzymatic activity of alkaline phosphatase (Brückner et al., 2000). S2 cells were transfected with the expression vectors for the proteins indicated as + or the empty control vectors to keep the total amount of the plasmids constant. The relative binding of the ligand was expressed as an arbitrary unit that takes the normalised bound AP activity of the empty vector-transfected cells as zero, and that of the cells transfected with Notch alone as one. Means±s.d. of triplicate assays are shown. Lower panel, Western blots of S2 cell extracts from the cells prepared in parallel to those used for the binding assays presented in the upper panel. The blot was probed with the anti-Notch, anti-Myc, anti-HA and anti-tubulin antibodies. Two forms of Notch were recognised by the anti-Notch antibody (9C6) that directs a region of the intracellular domain of Notch. The upper band is the unprocessed form of Notch and the lower band is the C-terminal part of the cleaved form. (B) Co-expression of nti-IR (RNAi for nti) with Notch abolishes the binding to Delta-AP irrespective of Fng overexpression. The analysis was carried out and the results were presented as in A. (C) Expression of Notch on cell surface is not influenced by the changes in Nti expression. The cells transfected with the indicated expression vectors were analysed by flow cytometry with an antibody directed against the extracellular domain of Notch (Rat-1). The numbers denote the percentages of cells in the indicated squares.

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