Purified c-Secretase Complexes with PS1 Mutations Figure 4. High-grade purification of human c-secretase complexes with FAD-linked PS1 mutants. (A) Schematic representation of the c- secretase purification process. Briefly, Presenilin double-knockout MEFs were used to first generate cell lines that stably overexpress human c-secretase complexes containing different PS1 variants. Next, these cell lines were used for a multi-step purification procedure as described in the material and methods. (B) Blue-Native PAGE analysis of purified c-secretase complexes made of different PS1 variants. Equal volumes of the different purified c-secretase preparations were separated by native-PAGE on a 4–16% Bis-Tris gel, and stained with silver nitrate (top panel), or immunostained for NCT (NCT164, middle panel) or PS1-NTF (ab10281, bottom panel) as indicated. c-Secretase complexes appeared on the gel as high molecular weight complexes (HMWCs) of ,350 kDa. Note that the levels of HMWCs were similar for all clones. (C) Equal volumes of purified c-secretase complexes with FAD-linked PS1 mutants were separated under denaturing conditions (SDS-PAGE) and immunostained with anti-NCT (NCT164), anti-PS1-NTF (MAB1563), anti-PS1-CTF (MAB5232), anti- HA (3F10), or anti-Flag (M2) antibodies. Two independent purifications were performed on each clone with similar results. A representative dataset is shown. doi:10.1371/journal.pone.0035133.g004 PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35133