Fig. 5. Requirement of the intracellular domain of Delta1 for full activation of N2. (A) Generation of D1Δ^ICD-CHO [CHO(r) cells expressing the truncated Delta1 lacking its intracellular domain]. To investigate the expression of fD1 and D1Δ^ICD, a cell-binding assay using sN1-Fc (6.7 nM) was performed against the fD1-CHO and D1Δ^ICD-CHO cells. (B) Comparison of signal-transducing activity of fD1 and D1Δ^ICD. To examine activity of the two molecules, a transient reporter assay with a TP1-luciferase reporter plasmid was performed using fN2-CHO cells. Fold-induction of luciferase activity for fD1-CHO and D1Δ^ICD-CHO (mean of triplicate measurements with standard deviation) was calculated against luciferase activity when parental CHO(r) was used as stimulator. (C) N2 fragments after fD1 and D1Δ^ICD stimulations. BaF3 was stimulated for 1.5 h under the conditions indicated in the figure and then separated into two fractions, membrane/cytosol-rich and nucleus-rich. In each fraction, N2 fragments containing an intracellular domain were analyzed by western blot using the bhN6 antibody after immunoprecipitation.