PS1 complexes are detected in human CSF with alternative antibodies. (A) Schematic representation of PS1 with epitopes for the anti-PS1 antibodies indicated. (B) Human ventricular post-mortem CSF samples from non-demented controls (NDC) were blotted under denaturing conditions with the indicated anti-PS1 antibodies. (C) Loss of CSF-PS1 reactivity in CSF samples heated at 98°C compared to 50°C using the NTF-PS1 antibody from Calbiochem. The stability of CSF-PS1 is also affected after 20 minutes of heating at 50°C resulting in loss of immunoreactivity. (D) CSF samples were also blotted for the homologue PS2 with an anti-CTF antibody (00/12) and for other subunits of the γ-secretase complexes, APH-1, PEN-2 and nicastrin. Nicastrin was the only γ-secretase partner with a different banding pattern, with a 130-kDa band corresponding to the molecular mass of the protein, which was weakly detectable in some of the CSF samples tested. The ~35-kDa band (*) may correspond to nonspecific binding by the antibody, as fragments of this size have not been previously demonstrated to be positive for nicastrin. (E) Effect of denaturation temperature prior to electrophoresis (heating for 5 min at 98°C or 10 min at 50°C) on stability of APH-1, PEN-2 and nicastrin. High molecular mass complexes of APH-1 and PEN-2 were only detected after heating at 50°C, while the 130 kDa-kDa nicastrin subunit was detected after denaturation at 98°C, indicating that nicastrin is not a part of the complexes. Illustrative examples (from 3 different experiments).