Since it has been established that Ab and AICD are generated following the processing by c-secretase at two distinct gamma- and epsilon-cleavage sites in APP [60], further investigation is now required to better understand whether and how FAD mutations in PS1 differentially affect the epsilon versus gamma-cleavage sites in APP, as well as in other known c-secretase substrates. Materials and Methods DNA Constructs and mutagenesis NCT-V5, Flag-PEN2, APH1aL-HA, wild-type PS1 human cDNAs (PS1-WT) were obtained from D. Selkoe (Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA). PS1 DE9 cDNA was obtained from C. Saura (Universitat Auto` noma de Barcelona, Spain). PS1 D257A and PS1 D385A cDNAs were obtained from M. Wolfe (Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA). Mutations in PSEN1, namely, L166P and P436Q, were generated by PCR-based mutagenesis on the plasmid pcDNA3.1/Zeo(+)-PS1-WT using T7 or BGH primers (specific to the plasmid DNA sequence) together with the corresponding primers: L166P Rv: 59 CAG CAA CAA Taa gct tGA TAT AAT AGG 39; L166P Fw: 59 CTT ATT ATA TCa agc ttA TTG TTG CTG 39; P436Q Rv: 59 TCG AGT TTA Gaa gct tTC TTG AAA ATG GCA AGG AG 39; P436Q Fw: 59 TCG AGT TTA Gaa gct tTG CCA GCT CTT CAA ATC TCC 39. The sequences corresponding to enzyme restriction sites are given in small letters. The PCR fragments were next digested with BamHI/HindIII and HindIII/XhoI respectively, and were subcloned into pcDNA3. PS1 D257A/D385A (DDAA) was obtained by subcloning the DraI/ XhoI digested fragment of PS1 D385A into pcDNA3.1-PS1 D257A. The APP based substrate SPA4CT-Flag, corresponding to the signal peptide of APP fused to the APP-C99-Flag with a small linker region (DA) [36], was obtained by removing the HindIII/EcoRI fragment of pcDNA3-APP695-Flag and inserting the following annealed primers: 59 AGC TTA TGC TGC CCG GTT TG GCA CTG CTC CTG CTG GCC GCC TGG ACG GCT CGG GCG GAT GCA GAT GCA G 39 and 59 AAT TCT GCA TCT GCA TCC GCC CGA GCC GTC CAG GCG GCC AGC AGG AGC AGT GCC AAA CCG GGC AGC ATA 39. All constructs were next subcloned into the self-inactivated vector pSIN-PGK-WHV cassette (a kind gift from R. Zufferey, Brain Mind Institute, EPFL, Lausanne, Switzerland). Inserts for plasmids pET21-C100-Flag, and pET21-C100-His were generated by PCR as previously described [27]. Plasmids pMD2G and psPAX2 were obtained from D. Trono (Global Health Institute, EPFL, Lausanne, Switzerland). Lentivector production Replication-defective lentiviral particles were produced by a three-plasmid transient transfection of cells from the human embryonic kidney HEK 293T cell line [61]. Briefly, cells were incubated in 10 cm dishes until they reached 70% confluence and were transiently co-transfected by the calcium phosphate method, with 5 mg of envelope plasmid (pMD2G), 15 mg of packaging plasmid (psPAX2), and 20 mg of vector plasmids (pSIN-PGK- WHV). Cells were incubated overnight with DNA-calcium precipitates, washed twice with Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and incubated with 7 mL of DMEM. After 24 h, the conditioned medium was harvested and cells were incubated a second time with 7 mL of DMEM for 24 h. Media were then pooled, passed through 0.45 mm filter, and stored at 280uC as 2 mL aliquots until use. A p24 ELISA Purified c-Secretase Complexes with PS1 Mutations (Zeptometrix corporation) was performed on each batch of media in order to evaluate the number of viral particles generated. Generation of stable cell lines through multiple gene transductions Mouse Embryonic Fibroblasts (MEFs, obtained from B. De Strooper, Flanders University Institute of Biotechnology, Belgium) defective in PSEN1 and PSEN2 [30,31] were cultivated in 10 cm dishes in DMEM, supplemented with 10% foetal bovine serum (FBS) and penicillin/streptomycin (P/S) (Invitrogen). A first set of stable cell lines was generated by co-transducing NCT-V5, APH1aL-HA, and Flag-PEN2 containing lentiviral vectors (LV) repeatedly for two weeks at each passage (1/20). Ten clones were isolated by the limiting dilution method and were further screened for NCT-V5, Flag-PEN2 and APH1aL-HA expression by immunodetection. The clone that showed the highest expression of the three proteins (designated c - PS1/2) was used as the parental cell line to generate the stable cell lines expressing different PS1 variants, by the same method and by using lentiviral vectors carrying human PS1-WT, PS1-L166P, PS1-DE9, PS1- P436Q, PS1-D257A, PS1-D385A, and PS1-DDAA. For each PS1 variant, five clones were selected for a full characterization and two of them were further used for c-secretase purification. For SPA4CT overexpression experiments, 500,000 cells of each clone were exposed to the same arbitrary dose of lentiviral vectors carrying human SPA4CT-Flag in 2 mL of DMEM, 1% FBS, P/S and were plated in 6-wells plates for 72 h. The medium was next replaced by fresh DMEM, 2% FBS, P/S for 24 h. Finally, cell culture medium was harvested, supplemented with protease inhibitor cocktail (Roche), centrifuged for 3 min at 1,0006 rpm and frozen at 280uC until further processing. Cells were washed in PBS and submitted to protein extraction. Protein extraction Total protein extracts were prepared in 1% NP40 - HEPES buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2), supplemented with protease inhibitor cocktail (Roche), and were clarified by centrifugation for 1 h at 16,0006 g, 4uC. Cell membranes were obtained as described below and solubilised in 1% CHAPSO - HEPES buffer and centrifuged at 16,0006 g for 1 h. Supernatants corresponding to the microsomal protein extracts were harvested and stored at 280uC. Protein content of the extracts was estimated by the BCA protein assay reagent kit (Pierce). Multi-step purification of human c-secretase complexes with FAD-linked PS1 variants 1) Cell membrane preparation. MEF PS1/PS2 DKO cells (1.06109 cells) expressing NCT-V5, Flag-PEN2, APH1aL-HA and different forms of PS1: wild type (WT), or with FAD-linked variants or lacking the critical aspartate residues, were collected from 15 cm dishes and fully resuspended in 40 mL of MES buffer (50 mM MES, pH 6.0, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2), supplemented with protease inhibitor cocktail (Roche). Next, cells were passed four times through a high-pressure homogenizer (Emulsiflex-C5, Avestin Inc, ON Canada) at a pressure greater than 1,000 psi. Nuclei and unbroken cells were removed by centrifugation at 3,0006 g for 20 min at 4uC in a Beckman Coulter Allegra X-15R centrifuge. The supernatant was collected and centrifuged at 100,0006 g for 1 h at 4uC in a SW32Ti rotor using a Beckman Coulter Optima L-80 ultracentrifuge to recover the membrane preparation in the pellet. 2) Bicarbonate wash. The membrane pellet was fully resuspended in 1.6 mL of ice cold PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35133