Purified c-Secretase Complexes with PS1 Mutations Figure 3. Enzymatic activity of partially purified c-secretase complexes with FAD-linked PS1 mutants. (A) c-Secretase activity assays performed with c-MEF and c - PS1/2 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). The relative amounts of AICD-Flag generated in the reactions, reflecting c-secretase activity, were estimated by densitometry. PS1 immunostaining was used to assess the amount of input material. (B) Equal amounts of microsomal proteins were immunoprecipitated overnight at 4uC with either anti-Flag M2 or anti-HA affinity resins, and PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35133