Potency of γ-secretase inhibitors in various activity assays. The potency of two structurally unique GSIs was assayed in four unique γ-secretase activity assays. IC50 values were determined from the dose response curves using a non-linear regression analysis in the Prism software. An in vitro assay was based on the one previously reported by Li et al. , except we utilized Sb4 substrate that eliminated the need for biotinylated antibody. The cell-based activity assay used N2A mouse neuroblastoma cells stably over-expressing APP and a biotinylated 4G8 antibody that binds the C-terminus of the β-amyloid peptide. The exo-cell assay incubated HeLa cells simultaneously with GSI, Sb4 substrate as well as 0.25% CHAPSO detergent prior to detecting substrate cleavage. Finally, the extended exo-cell assay first incubated HeLa cells with GSI for 24 hours. Subsequently, the cells were washed 1× in PBS and then incubated with Sb4 substrate and CHAPSO detergent. The assay was then carried out exactly as described for the original exo-cell method. All assays incorporate ruthenylated G2-10* antibody to detect 40-site cleavage of APP or recombinant Sb4 substrate and quantitated activity by measuring ECL.