Tricine sds running buffer

Forty-eight hours post transfection, the HEK293 cells were lysed using CelLytic M lysis reagent (Sigma-Aldrich). Clarified cell lysates (30 μl) were mixed with 2× Tricine SDS Sample Buffer (Novex, Thermo Fisher Scientific) and run on 10–20% Tricine Protein Gels (Novex, Thermo Fisher Scientific) in Tricine SDS Running Buffer (Novex, Thermo Fisher Scientific) at 125 V for 90 min. Proteins were transferred to polyvinylidene fluoride membrane (0.2 μm, Bio-Rad) at 100 mA for 2 h in Tris-Gly Transfer Buffer (Novex, Thermo Fisher Scientific) supplement with methanol (Sigma-Aldrich). Immunoblots were incubated with primary monoclonal anti-FLAG M2 antibody (1:1000, F1804-200UG, Sigma-Aldrich) and anti-β-actin (1:10,000, AM4302, Ambion, Thermo Fisher Scientific) overnight at 4 °C and then secondary anti-mouse IgG and horseradish peroxidase-linked antibody (1:5000, Cell Signaling) at room temperature for 2 h. Immunoblots were developed with Western ECL (Clarity, Bio-Rad). Full, uncropped versions of all blot images are provided in Supplementary Fig. 16.

Aβ₄₂ aggregate samples were diluted with Tricine SDS Sample Buffer (Cat # LC1676, Thermo Fisher Scientific, Waltham, MA, USA) and separated on a 10–20% Tris-Tricine gel using Tricine SDS Running Buffer (Cat # LC1675, Thermo Fisher Scientific, Waltham, MA, USA). The separated bands were transferred onto a nitrocellulose membrane and detected with the mouse monoclonal anti-Aβ oligomer specific antibody NU-2 (1:4000; Klein’s lab) and the mouse monoclonal anti-β-Amyloid antibody 6E10 (1:1000; Cat # 803001, BioLegend, San Diego, CA, USA). The membrane was probed with s680RD Goat anti-Rabbit IgG Secondary Antibody (1:10,000; Cat # 926-68071, LI-COR Biosciences, Lincoln, NE, USA) and 800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000; Cat # 926-32210, LI-COR Biosciences, Lincoln, NE, USA). The protein bands were quantified by the Image Studio Lite software (version 5.2, LI-COR Biosciences, Lincoln, NE, USA). Molecular weights were estimated using a prestained protein ladder from Bio-Rad (Hercules, CA, U.S.A.).