Xt mes running buffer

The eluted proteins were precipitated with trichloroacetic acid (TCA) to enrich them for subsequent analyses. Sodium deoxycholate was added to a final concentration of 0.02%. Samples were mixed, placed on ice and supplemented with TCA to a final concentration of 10%, and then incubated on ice for 1 h. Precipitated proteins were pelleted by centrifugation (10 min, 16000 × g, 4°C), washed with 100% ice-cold acetone by incubating them on ice for 15 min and subsequent centrifugation (10 min, 16000 × g, 4°C). Washing was repeated once; the pellet was air-dried and boiled in SDS-PAGE buffer (7.5 μl 4x XT Sample Buffer, 1.5 μl 20x XT Reducing Agent, 1% SDS, ad 30 μl H2O) for 5 min at 95°C. The samples were separated on Criterion XT Bis-Tris Precast Gels (125 V for 1.5 h in XT MES Running Buffer) (all from Bio-Rad Laboratories GmbH). PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a size standard. For a subsequent Coomassie staining, the gel was equilibrated in H₂O for 5 min, incubated in 100 ml acetic acid/ethanol/H₂O (10:40:50) for 1 h and subsequently rehydrated with H₂O (three times, 10 min). The gel was stained with PageBlue^TM Protein Staining Solution (Thermo Fisher Scientific) overnight and repeatedly washed with H₂O for destaining.

For SDS-PAGE analysis, the pellets containing the cytoplasmic contents were treated with CelLytic™ B Cell Lysis Reagent (Sigma-Aldrich). An additional 50 units/mL benzonase nuclease (Sigma-Aldrich) was used for all samples. The samples were incubated for 30 min at room temperature with shaking (100 rpm). Dilutions (5x) of the samples were done with XT MES Running Buffer (Bio-Rad). Each of the samples was combined with XT Sample Buffer 2X (Bio-Rad) and with XT Reducing Agent (Bio-Rad) and incubated at 95 °C for 5 min. Samples (10 μL, 10x diluted) and a ladder (Precision Plus Protein™ Dual Color Standards, Bio-Rad, 5 μL) were loaded on SDS-gel (Criterion™ XT Bis-Tris Precast Gels, 12%, Bio-Rad) for gel electrophoresis (200 V, 45 min), which after end of run was stained with InstantBlue™ Coomassie Protein Stain (Expedeon).

Cells or subcellular components were lysed three times with 1× cell lysis buffer (Cell Signaling Technology, 9803) containing protease inhibitor on ice for 10 min. Protein was quantified using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, 23225), and 20 to 40 µg of each sample was resolved on 4 to 12% Criterion XT Bis-Tris gels (Bio-Rad, 3450124) in XT MES running buffer (Bio-Rad, 1610789) and transferred to polyvinylidene difluoride membranes (Bio-Rad, 1620233) using the Trans-Blot Turbo Transfer Pack and System. Membranes were blocked with tris-buffered saline with Tween 20 (TBST) containing 5% skim milk for 1 h and incubated overnight at 4 °C with various primary antibodies. Following three washes in TBST, membranes were incubated with goat anti-rabbit or anti-mouse immunoglobulin G (IgG) horseradish peroxidase secondary antibody (Cell Signaling Technology, 7074 or 7076; 1:1000) at room temperature for 1 h and washed. Chemiluminescence substrate was applied using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095), and blots were analyzed using the ChemiDoc Touch Imaging System (Bio-Rad) and Image Lab Software (Bio-Rad, version 6.1)^{77 (link)}. The information on antibodies is shown in Supplementary Table 1.

For western blot, samples were diluted in LDS (Lithium Dodecyl Sulfate) sample buffer (NuPage) and sampling reducing agent in order to load 20 µg of proteins. After heating, samples were loaded on a 4–12% Criterion™ XT Bis–Tris gel (Bio-Rad), migrated in XT MES Running Buffer (Bio-Rad) for 1 h at 110 V and transferred onto 0.2 µm nitrocellulose. For dot blot, 2µL of samples were directly loaded onto 0.2 µm nitrocellulose. After 1 h of blocking at room temperature, membranes were blotted overnight at 4 °C with primary antibodies 6E10 (Against human Aβ_1-16; Biolegend), APP-Cter-17 (against the last 17 amino acids of the human APP sequence [39 (link), 51 (link)], A11 (against oligomeric species, ThermoFisher) and OC (anti-amyloid fibrils, Rockland). After rinse in TBS-T, membranes were incubated with secondary antibodies for 1 h at room temperature. Proteins were revealed using horseradish peroxidase (HRP) and ECL™ Western Blotting Detection Reagent (G&E Healthcare). Quantifications of protein expression levels were performed on ImageJ Software.