Vivaspin 6

The βPFO_Aβ42/DPC complex was trapped in NAPols using 1:2, 1:4, and 1:8 (w/w) Aβ42/NAPol ratios following the same protocol as described in section “Selection of the most suitable type of APol for βPFO_Aβ42trapping.” After DPC removal, the samples were incubated for 24 h at 37°C in order to determine the stability of the βPFO_Aβ42/NAPol complex. Only when indicated in the paper and when more extensive detergent removal was required, after DPC removal with Biobeads, three additional dilution/concentration steps were performed. These consisted of a 10-fold dilution of the βPFO_Aβ42/NAPol complex by addition of a 10 mM Tris·HCl pH 9.0 solution with 10% D₂O, followed by a 10-fold concentration of the resulting solution using a Vivaspin 6 (Sigma) centrifugal concentrator device (MWCO 5000 Da). The concentration steps were carried out at 4°C.

Total protein extracted in a lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 1 mM EDTA) containing protease/phosphatase inhibitors (Sigma-Aldrich) was separated by SDS-PAGE and transferred onto Hybond-ECL membrane (Amersham) before being probed with primary antibodies diluted in blocking buffer (5% skim milk in TBS with 0.05% Tween 20 (TBST)). After washing in TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies before analyses by enhanced chemiluminescence (GE Healthcare). Co-immunoprecipitation was performed using pre-cleared lysate (1–2 mg protein) and the immune complexes captured on protein A/G-Agarose beads (Santa Cruz Biotechnology) were analysed by Western blot. Conditioned medium was collected from NIH3T3 cultured in 0.5% serum-containing medium for 48 h with or without TMI005 (Sigma-Aldrich) and concentrated using Vivaspin 6 with 10 kDa MWCO (Sigma-Aldrich).