Bestain

Cell lysis and Western blotting were performed as described previously [19 (link),41 (link)]. Detergent lysates of cells were prepared using RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with protease inhibitors (a mixture of 4-(2-aminoethyle) benzenesulfonyl fluoride, pepstatin A, E-64, bestain, leupeptin and aprotinin; Sigma). For sequential extractions, cells were lysed with 1% Triton X-100 in PBS and then the pellets were resuspended and lysed in 2% SDS. The bicinchoninic acid (BCA) assay was performed to quantitate total protein in accordance with the manufacturer’s instructions (Millipore). Lysates were subjected to 4–2% Bis-Tris SDS-PAGE and transferred to a nitrocellulose membrane prior to incubation with selected antibodies. Immunoreactive bands were visualized using an Odyssey infrared scanner (LiCor Biosciences).
The antibodies used in this study were αSyn (610786, BD); αSyn (AB5038; Millipore); pSer129-αSyn (81A)[42 (link)]; PAN-Syn (AB6176, Abcam); RER1 (AP-R76) [19 (link)] and (R4407 for endogenous RER1, Sigma); β-actin (AC-15, Sigma); GAPDH (14C10, Cell Signaling); Flag (M2, Sigma); HA (H3663, Sigma); GM130 (610823, BD); KDEL (10C3, Stressgen); APP (A8717, Sigma); EGFP (A11122, Life Technologies).

BACE1 (human recombinant β-secretase) (EC 3.1.1.8) and the BACE1 FRET (fluorescence resonance energy transfer) assay kit Red were purchased from ThermoFisher Scientific (P2985; Waltham, MA, USA). Electric eel AChE (EC3.1.1.7), horse serum BChE (EC 3.1.1.8), acetyl thiocholine iodide, butyryl thiocholine chloride, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), quercetin, berberine, and the MTT assay kit were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). For the cell culture experiment, Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany). β-secretase inhibitor III (β-SI) was purchased from Calbiochem (Darmstadt, Germany). The protease inhibitor mixture (a mixture of AEBSF, pepstatin A, E-64, bestain, leupeptin, and aprotinin) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for Aβ40 and Aβ42 were obtained from IBL (Kunma, Japan). All chemicals and solvents used were reagent grade, purchased from commercial sources, and used as received.