Characterization of CSF-PS1 complexes. (A) Brain γ-secretase complexes (frontal cortex from non-diseased control subjects extracted in buffer containing 0.5% dodecylmaltoside) were analyzed by blue native-PAGE and compared with PS1 complexes isolated from lumbar CSF samples (NDC cases). Incubation of blots with antibodies for the different γ-secretase subunits confirmed that nicastrin is not present in CSF-PS1 complexes. (B) The same brain extract and CSF samples were also fractionated on 5-20% sucrose density gradients. The fractions (collected from the top of each tube) were immunoblotted for NTF-PS1 under denaturing conditions with the antibody from Calbiochem. Enzymes of known sedimentation coefficient, β-galactosidase (G, 16.0S; ~540 kDa), catalase (C, 11.4S; ~232 kDa) and alkaline phosphatase (P, 6.1S; ~140-160 kDa) were used as internal markers. (C) CSF fractions from sucrose gradients were also blotted for APH-1, PEN-2 and nicastrin. Nicastrin was mostly undetectable in fractionated CSF samples.