FIGURE 2.

In vitro γ-secretase activity for proteolysis of αCTF and βCTF. A, schematic representation of MBP-βCTF and MBP-αCTF. The N and C termini of αCTF and βCTF are tagged with MBP and FLAG, respectively. There is a thrombin cleavage site between MBP and the target proteins. The cleavage sites of thrombin and α- and γ-secretase are indicated by arrows. The recognition epitope of antibodies that have been used in the assay of D are indicated. B and C, analyses of αCTF and βCTF. Purified proteins were separated by SDS-PAGE and stained by Coomassie Blue (B). The protein masses were determined by LC-MS (C). The molecular masses of αCTF and βCTF were calculated through deconvolution of the mass-to-charge ratio (m/z). D, in vitro γ-secretase activity for X40 production from αCTF and βCTF substrates. Each substrate was incubated with 4 μg of HeLa membrane at 0.1, 0.5, and 1 μm in the presence of 0.25% CHAPSO. After a 2.5-h incubation, the reaction was stopped by adding radioimmuneprecipitation assay buffer, and the product X40 was assayed with biotinylated 4G8 and ruthenylated G2-10 antibodies by ECL (mean ± S.E. (error bars), n > 3). The amount of X40 was determined using synthetic Aβ40 and P3 peptides as standards. Note: in all figures, the assay background was defined in the presence of 1 μm L-685,458 for in vitro assays. γ-Secretase activity was calculated by subtracting the assay background from the signal that was detected in the absence of inhibitor (dimethyl sulfoxide only).