*3.4. Western Blot of Acetylated Tau*

Tau acetylation was verified by western blot against acetyl-lysine. 100-ng samples of Ac-Tau, wt Tau and BSA were loaded on a 4–20% gradient SDS-PAGE gel (Mini-PROTEAN TGX Precast Gels, Bio-Rad). After electrophoresis, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System, following the manufacturer's protocols (Bio-Rad). After incubation with 5% (*w*/*v*) nonfat milk in TBS-T (150 mM NaCl, 0.1% Tween-20, 20 mM Tris-HCl, pH 7.5) at RT for 2 h, the membrane was incubated with antibody against acetyl-lysine (1:100 in 1% nonfat milk/TBS-T; sc-32268, Santa Cruz Biotech, Dallas, TX, USA) overnight at 4 ◦C. The membrane was washed six times for 10 min with TBS-T and incubated with HRP-conjugated anti-mouse antibody (1:1000; #7076, Cell Signaling, Danvers, MA, USA) at RT for 30 min. The membrane was washed six times and developed with Clarity Western ECL Substrate according to the manufacturer's protocols (Bio-Rad). Chemiluminescent signals were measured using ChemiDoc MP Image System (Bio-Rad).
