Chlorpromazine

When treating cells with chlorpromazine or Filipin, cells were first incubated with 10 µg/ml chlorpromazine (Sigma) or 5 µg/ml Filipin (Sigma) in 10% FBS McCoy’s 5 A medium for 30 min. After that, the cells were incubated with 4 μM PV-1 and 5 μM Tubulin-FITC for 1 h. Then cells were washed with PBS and incubated in 10% FBS fresh medium for 1 h.
When treating with EIPA (5-(N-ethyl-N-isopropyl) amiloride), cells were incubated with 100 μM EIPA, 4 μM PV-1, and 5 μM Tubulin-FITC for 1 h. After that, the cells were washed with PBS and incubated in cell culture medium 10% FBS for 1 h.
After the above treatment, cells were imaged by confocal microscopy or analyzed by microcapillary flow cytometer (Guava easyCyte 8HT, EMD Millipore Corporation).

The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich). Except for brefeldin A and chlorpromazine, which were diluted in ethanol or water respectively, all stock solutions were prepared using dimethyl sulfoxide (DMSO, #276855, Sigma-Aldrich). Cells were preincubated at 37°C and 5% CO₂ with the specific inhibitors diluted in HBSS for 60min (cytochalasin D, latrunculin A) to 100min (all others) and were present during all functional analyses.

Various inhibitors were used to study the transport mechanisms of Aβ and its isoforms. The contribution of RAGE to the transport of Aβ across the endothelial monolayer was assessed using the antagonist of this receptor FPS-ZM1 (Sigma) at a concentration of 20 μM (to obtain a stock solution, FPS-ZM1 was dissolved in DMSO to a concentration of 305 mM). To study the caveolin-dependent transport of Aβ isoforms, the inhibitor filipin (Sigma) was used at a concentration of 3 μg/ml (to obtain a stock solution, filipin was dissolved in DMSO to a concentration of 5 mg/ml). An equivalent amount of DMSO was added to the control samples. The contribution of clathrin-dependent endocytosis was assessed using chlorpromazine (Merck) at a concentration of 5 μg/ml (to obtain a stock solution, chlorpromazine was dissolved in DMEM). These concentrations were selected based on literature data and tested for toxicity to bEnd.3 cells using MTT (filipin) and WST (FPS-ZM1 and chlorpromazine) assays according to the manufacturer's protocol (Supplementary Figure 2). Before experiments, cells were preincubated with inhibitors added to the upper transwell compartment for 1 h, after which they were filled with solutions containing the inhibitor and 1 μM Aβ, and samples were taken from the lower compartment after 2, 6 and 24 h.