sodium bicarbonate buffer (0.1 M NaHCO3, pH 11.3) by pipetting up and down at least 30 times, and incubated at 4uC for 20 min. The washed membranes were then centrifuged at 100,0006 g for 1 h at 4uC and stored at 280uC until use. 3) Solubilisation of c-secretase complexes. The bicarbonate-washed mem- branes were fully resuspended in 1.7 mL of 1% CHAPSO - HEPES buffer by pipetting up and down at least 30 times. The membranes were then incubated at 4uC for 1 h. To pellet the insoluble material, the solution was centrifuged at 16,0006 g at 4uC for 1 h, the pellet was discarded, and the supernatant saved. This lysate is defined as ‘‘solubilised c-secretase preparation’’. Next, these freshly prepared solubilised preparations were used for affinity purification of c-secretase complexes as described below. 4) Anti-Flag M2 affinity purification. The solubilised c-secretase preparations were first diluted 1:2 with HEPES buffer, and further diluted 1:6 with 0.1% digitonin - TBS buffer (50 mM Tris- HCl, pH 7.4, 150 mM NaCl) (final detergent concentration: 0.08% CHAPSO, 0.08% digitonin) and incubated overnight at 4uC with agitation after adding 200 mL of anti-Flag M2 affinity resin beads (Sigma-Aldrich) that had been pre-equilibrated in 0.1% Digitonin - TBS buffer. The beads were washed 3 times in the same buffer, and the bound proteins were eluted over 4 h at 4uC with 0.4 mL of this buffer containing 200 mg/mL of Flag peptides (Sigma-Aldrich). This elution step was repeated once for 1 h at 4uC and the eluted fractions were pooled and designated ‘‘M2 pooled 800 mL fractions’’. 5) Anti-V5 affinity purification. The M2 pooled fractions (volume made up to 1 mL with 0.1% Digitonin - TBS buffer) were next incubated overnight at 4uC under agitation with 200 mL of anti-V5 affinity resin beads (Sigma- Aldrich), pre-equilibrated in 0.1% digitonin-TBS buffer. The beads were washed 3 times in the same buffer, and the bound proteins were eluted for 1 h at 4uC with 0.4 mL of this buffer containing 500 mg/mL of V5 peptides (Sigma-Aldrich). This elution step was repeated 4 times, and the eluted fractions were pooled and designated ‘‘V5 pooled 1.6 mL fractions’’. 6) Anti-HA affinity purification. As a final purification step, the V5 pooled fractions (volume made up to 1.8 mL with 0.1% digitonin - TBS buffer) were next incubated overnight at 4uC under agitation with 200 mL of anti-HA affinity resin beads (Sigma-Aldrich), pre- equilibrated in 0.1% digitonin - TBS buffer. The beads were washed 3 times in the same buffer, and the bound proteins were eluted overnight at 4uC with 0.2 mL of this buffer containing 200 mg/mL of HA peptides (Sigma-Aldrich). This elution step was repeated once, for 1 h at 4uC, and the two eluted fractions were kept separately, designated ‘‘HA fraction E1’’ and ‘‘HA fraction E2’’. Co-immunoprecipitation Co-IPs were performed on microsomal protein extracts. Briefly, 100 mg of proteins were diluted in a final volume of 1 mL of 1% CHAPSO - HEPES extraction buffer containing protease inhibitors (Roche), and 50 mL of pre-equilibrated anti-Flag or anti-HA affinity beads (Sigma-Aldrich) were added. Samples were next incubated overnight at 4uC on a rotator wheel and were washed three times in 0.2% CHAPSO - HEPES buffer. Finally, beads were resuspended in 50 mL of 0.2% CHAPSO - HEPES buffer and were used for in vitro c-secretase assays. In vitro c-secretase activity assays In vitro c-secretase activity assays were performed as previously described [27]. c-Secretase APP-based substrates were expressed in E. coli transfected with plasmids pET21-C100-Flag or pET21- C100-His as a fusion protein consisting of a Met for translation initiation, and either the Flag tag sequence (C100-Flag) or the His Purified c-Secretase Complexes with PS1 Mutations tag sequence (C100-His), and were affinity purified using an anti- Flag resin (M2, Sigma-Aldrich) or an Ni-NTA agarose resin (Invitrogen), respectively. In vitro assays were performed at 37uC for 4 h, with 1 mM of recombinant substrate, 0.025% phospha- tidylethanolamine (PE) and 0.1% phosphatidylcholine (PC). c- Secretase activity was quantified by measuring the amount of AICD and Ab generated during the reaction, by immunoblot or sandwich ELISA respectively, as described below. SDS-PAGE, Native PAGE, Western blotting and antibodies Total or microsomal protein extracts were resolved by electrophoresis on NuPAGEH NovexH 4–12% Bis-Tris Gels (Invitrogen) or on standard 12% acrylamide/bisacrylamide Tris Glycine gels for SDS-PAGE analysis. Purified c-secretase was resolved by electrophoresis on NativePAGETM NovexH Bis-Tris 4–16% Gels for Blue Native (BN)-PAGE analysis (Invitrogen). Silver staining was performed directly on gel according to manufacturer instructions (Biorad). For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (What- man), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma- Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for b- actin (Sigma-Aldrich). Anti-mouse/rabbit/rat IgG conjugated to Alexa 680 were purchased from Invitrogen. The Odyssey infrared imaging system (LICOR) was used to detect the fluorescent signal. Quantification of Ab peptides Ab peptides from the c-secretase assays described above were quantified by sandwich ELISA according to the protocol provided by the manufacturer. Three kits were used to detect human Ab1– 40 (Invitrogen KHB3481) and Ab1–42 (Invitrogen KHB3544 and Wako 269-64401). Supporting Information Figure S1 Characterization of stable cell lines overex- pressing human FAD-linked PS1 variants in MEF PS1/ 22/2. Presenilin double-knockout mouse embryonic fibroblasts were stably transduced with lentiviral vectors carrying genes encoding hPS1 variants harbouring FAD-linked mutations or mutations in the catalytic aspartate residue(s), or PS1-WT, and cloned. Two clones per PS1 variant were selected for character- ization. (A) Whole cell protein extracts of the different cell lines were prepared in 1% NP40-HEPES buffer, separated by SDS- PAGE on 12% Tris-Glycine gels and analysed by immunostaining with various antibodies to detect the endogenous c-secretase core components: NCT (NCT164), hPS1 (NTF, MAB1563), and PEN2 (UD1), and with an antibody for APP (A8717). b-Actin was used as a loading control. Each lane represents one selected clone. (B) c- Secretase activity assays were performed with microsomal extracts prepared in 1% CHAPSO-HEPES buffer. Equal protein levels from the different extracts were diluted to 0.25% CHAPSO- HEPES buffer and incubated for 4 h at 37uC with lipids and 1 mM of recombinant human APP-based substrate (C100-Flag). Samples were analyzed by SDS-PAGE and immunostained with anti-Flag (M2) or anti-PS1 (MAB1563). PS1 immunostaining was used to assess the amount of input material. * Indicates a non-specific band, which was not detected in microsomal protein extracts of the same cell lines using the same antibody (MAB1563) (B). CTF: C-terminal fragment, FL: full-length, im.: immature NCT; m.: PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35133