One possible explanation for the increased Aβ production in the Aph1 L30F / T164A mutant is that the mutations increase complex formation . To test whether the Aph1 L30F / T164A mutants form a complex with other components , γ - secretase in yeast microsomes was immunoprecipitated with a PS1 antibody against the loop region ( GIL3 ) ( Figure 5 ) . NCT , PS1 , and Aph1 were coimmunoprecipitated with wild - type Aph1 or the . Aph1 mutations activate γ - secretase in yeast microsomes . Microsomes were prepared from yeast transformants expressing Aph1 WT or Aph1 L30F / T164A mutants , PS1 , NCT , FLAG - Pen2 , and the C55 fragment of APP ; they were then subjected to γ - secretase assays . CHAPSOsolubilized microsomes ( 40 µg protein ) were incubated at 37 • C for 24 h in the presence or absence of phosphatidylcholine ( PC ) ( 0.1 % ) and γ - secretase - specific inhibitor ( L685 , 458 ) . ( a , b ) Total Aβ production was analyzed by immunoblotting using the antibody 82E1 . ( c , d ) Aβ species were separated by urea / SDS - PAGE and analyzed by immunoblotting with 82E1 . Synthetic Aβ38 , Aβ40 , Aβ42 , and Aβ43 ( 30 pg ) were loaded as controls in the left - most lanes ( a , c ) . Amount of Aβ ( b , d ) and the ratio of Aβ species ( e ) were quantified from three independent assays ; data are shown with standard deviations . Statistical analyses were performed using one - way ANOVA followed by Dunnett 's multiple comparison test . Asterisks on each bar indicate p < 0.01 ( * * ) or p < 0.05 ( * ) with respect to Aph1 WT ( b , d , e ) . l. Sci . 2022 , 23 , x FOR PEER REVIEW Figure 5 . Aph1aL L30F / T164A mutant forms γ - secretase complexes in ye pared from yeast transformants as in Figure 4 and solubilized with buffe The γ - secretase complex was purified from extracts using an antibody a ( G1L3 ) or rabbit pre - immune serum , as indicated . Immunoprecipitants a analyzed by immunoblotting using specific antibodies . The input represe extract .