Purified c-Secretase Complexes with PS1 Mutations Figure 1. Generation of stable cell lines overexpressing all human c-secretase components with FAD-linked PS1 variants. MEF PS1/ 22/2 were stably co-transduced with lentiviral vectors carrying genes encoding hNCT-V5, Flag-hPEN2, hAPH1aL-HA and clones were isolated by limiting dilution to generate a cell line, designated as c- PS1/2, that overexpresses high amount of the three subunits. c- PS1/2 MEFs were further transduced with hPS1 variants harbouring FAD-linked mutations or mutations in the catalytic aspartate residue(s), or PS1-WT, and cloned. Each clone, derived form the c- PS1/2, was conveniently named according to the mutation present in PS1 preceded by the symbol c and followed by the number of the clone (c-MEF) in order to distinguish them from wild-type MEF (WT MEF) and MEF PS1/22/2. Two clones per c-secretase variant were selected for characterization. (A–B) Whole cell protein extracts of the different cell lines were prepared in 1% NP40-HEPES buffer, separated by SDS-PAGE on 4– 12% Bis-Tris or 12% Tris-Glycine gels and analysed by immunostaining to detect the c-secretase core components NCT (NCT164), PS1 (NTF, MAB1563; CTF; MAB5232), APH1aL-HA (3F10), and Flag-PEN2 (M2) (A), and endogenous APP (A8717) (B). b-Actin was used as a loading control. Each lane represents one selected clone. CTF: C-terminal fragment, FL: full-length, im.: immature NCT; m.: mature NCT, N: N-glycosylated, NTF: N-terminal fragment, O: O-glycosylated. doi:10.1371/journal.pone.0035133.g001 accumulation was similar in presence or absence of other human components of the c-secretase. Next, we further investigated the effect of PS1 variants on the cellular production/secretion of Ab species. For this purpose, and as described elsewhere [36], we used an APP-based c-secretase substrate with a Flag tag at its C-terminus (SPA4CT-Flag). This substrate was transduced in the above-described c-secretase overexpressing stable cell lines (c-MEFs) as well as in wild-type MEFs, and APP-CTFs and Ab levels were measured in cell lysate and cell media, respectively (Figure 2). Under these conditions, the overexpression of SPA4CT-Flag led to an accumulation of two bands corresponding to the exogenous substrate, associated with a concomitant decrease of endogenous APP-CTFs levels (Figure 2A and Figure S2). As estimated by ELISA, a three-fold increase in Ab1–40 and Ab1–42 levels was observed in the cell culture media of cell lines overexpressing human c-secretase (c-MEF WT), in comparison to untransduced WT MEFs (Figure 2B). Also, the average Ab1–42/Ab1–40 ratios were not significantly different in those cell lines (Mean 6 SD: c-MEF WT#5: 0.3060.01; c-MEF WT#8: 0.2960.01; WT MEF: 0.1960.06). Next, overexpression of SPA4CT-Flag in c-MEFs led to the pronounced intracellular accumulation of APP-CTFs in cells overexpressing the PS1 aspartate mutants (Figure 2C), and to the secretion in the cell culture media of different Ab levels as measured by ELISA (Figure 2D). First, only traces of both Ab1–40 and Ab1–42 were detected in c-MEFs overexpressing PS1 with aspartate mutants. Next, c-MEFs overexpressing PS1 with FAD-linked mutations displayed a pronounced variability in Ab levels when compared to PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35133