The coimmunoprecipitated α–γ-complex contains both α- and γ-secretase proteolytic activities. (A) S20 lysates were immunoprecipitated for A10, PS1, or just PAA. The immunoprecipitates were subjected to a γ-secretase activity assay (see Activity assays in Materials and methods) using a C100-Flag APP substrate. γ-Secretase activity was documented by generation of both AICD and Aβ via WB (left). The AICD generation was quantified (right). (B) S20 lysates were immunoprecipitated for γ-secretase using M2 (to Flag-Pen2) or 3F10 (to Aph-1-HA) and as controls TFR or M2 preabsorbed with Flag peptide. α-Secretase activity assays were performed on the resin using a fluorogenic peptide substrate (left) and quantified (right). A one-way ANOVA with Tukey’s posttest for multiple comparisons was used. *, P < 0.05. n = 6. After the activity assays, the immunoprecipitates were eluted and blotted for coIP of A10, NCT, and PS1 NTF. Error bars indicate the SD. im, immature; m, mature.