Generation of NICD and Aβ from NotchΔE and APP expressing cells. A. Twenty four hrs after the construct carrying NotchΔE was transfected into APP expressing HEK293 cells, cells were treated with DAPT or cpd E for 8 hr and lysed for WB with antibody 1744 to specifically detect the N-terminus of NICD. Bottom panel, the antibody against α-tubulin was applied to normalize the amounts of lysates used for WB. B. Levels of NICD determined by WB were quantified by densitometry (dotted line). Levels of Aβ generated from the γ-secretase cleavage of APP in the presence of DAPT were determined by ELISA. Comparison of NICD and Aβ generation in the presence of DAPT suggests that high concentrations of DAPT had a greater inhibition of Aβ than NICD. C. NICD (dotted line) and Aβ (solid line) production from cpd E-treated cells were compared. Cpd E inhibited Aβ generation with an IC50 of ~8 nM, and it shows a greater inhibition of Aβ than NICD. D. A luciferase reporter construct driven by HES1 promoter was transfected into HEK293 cells followed by treatment with cpd E or DAPT. Both γ-secretase inhibitors blocked transcriptional activation of NICD dependent luciferase activity.