Purified c-Secretase Complexes with PS1 Mutations Figure 2. Ab production in cell lines overexpressing human c-secretase components with FAD-linked PS1 variants. WT MEF, c-MEF and c - PS1/2 were transduced with an APP-based substrate corresponding to the 99 C-terminal residues of human APP fused to the APP signal peptide in N-terminus (SPA4CT [36]) and a Flag Tag in C-terminus. Cell proteins were extracted in 1% NP40-HEPES buffer, separated by SDS-PAGE on 12% Tris-Glycine gels and analysed by immunostaining with an antibody targeting the C-terminal part of APP (A8717) (A, C). Ab1–40 and Ab1–42 levels were also measured in the corresponding cell culture media (B, D). Data corresponds to three independent experiments (Mean 6 SEM). doi:10.1371/journal.pone.0035133.g002 transgenic animals with FAD-linked PS1 mutations show increas- ing brain levels of Ab1–42 [15,38]. Since Ab1–42, the first Ab specie deposited in the brain of AD patients [16,39], is more prone to aggregation when compared to shorter Ab species [40,41,42], it has been implicated in the seeding of amyloid plaques in AD patients with PSEN1 mutations [43]. This was further validated in vivo as the overexpression of PS1 mutants in APP transgenic mice accelerated the rate of Ab accumulation and deposition in the brain [44,45]. However, Bentahir and colleagues challenged this view by showing that several PSEN1 mutations were also capable PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35133