Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes
Figure 4
High-grade purification of human γ-secretase complexes with FAD-linked PS1 mutants.
(A) Schematic representation of the γ-secretase purification process. Briefly, Presenilin double-knockout MEFs were used to first generate cell lines that stably overexpress human γ-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 γ-secretase complexes made of different PS1 variants. Equal volumes of the different purified γ-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. γ-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 γ-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.