Generation of NICD and Aβ from purified APP and Notch substrate C100 and N100 in an in vitro γ-secretase activity assay. The E. coli generated APP- and Notch-based, 100-residue γ-secretase substrates C100-Flag and N100-Flag were mixed with the membrane vesicles solubilized in CHAPSO after DMSO or compounds were added. The mixture was incubated at 37°C for 4 hours. A. A dose-dependent inhibition of NICD generation by DAPT. Generation of NICD was detected by Western blot (WB) with antibody 1744 specifically recognizing N-terminus of NICD. B. Generation of NICD was inhibited in the presence of 100 nM of cpd E. C. Levels of NICD determined by WB were quantified by densitometry (dotted line). Levels of Aβ generated from the γ-secretase cleavage of C100 in the presence of DAPT were determined by ELISA (solid line). Comparison of NICD and Aβ generation in the presence of DAPT suggests that high concentrations of DAPT were more potent in blocking Aβ than NICD generation. D. NICD (dotted line) and Aβ (solid line) generation in the presence of cpd E were compared. Cpd E inhibited Aβ generation with an IC50 of 1 nM and is more potent in inhibiting Aβ than NICD.