Tau46

Total homogenates and fractionated tissue extracts were dissolved in SDS-sample buffer containing β-mercaptoethanol (2.5%). The heat-treated samples (55°C for 15 min) were separated by gel electrophoresis on 10% tris-glycine SDS–polyacrylamide gel electrophoresis gels (Nakarai, Japan) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). After blocking with a blocking solution containing 5% nonfat milk and 0.05% Tween 20 in TBS, the membranes were incubated with primary antibodies, such as Tau12 (provided by P. Davies, Feinstein Institutes for Medical Research), Tau46 (no. 4019, Cell Signaling, Danvers, MA), AT8 (MN1020, Thermo Fisher Scientific, Waltham, MA), LC3 (CAC-CTB-LC3-1-50, Cosmo Bio, Tokyo, Japan), and β-actin (A1987, Merck). After washing with TBS–Tween 20, the membranes were incubated with peroxidase-conjugated goat anti-rabbit antibodies (1:5000; Jackson ImmunoResearch, West Grove, PA) or anti-mouse immunoglobulin G (1:5000; Jackson ImmunoResearch). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer). Western blot immunoreactivity was visualized by Amersham Imager 600 (GE Healthcare). Quantitative analysis was performed with Image Studio Lite software (LI-COR, Lincoln, NE).

We used the following antibodies in this study: Tau46 (used in Figures 8, 9B; 1:1,000, #4019, Cell Signaling Technology, Danvers, MA, United States); Tau (used in Figures 1A, 9C, 1:1,000, #46687, Cell Signaling Technology, Danvers, MA United, States); CP13 Tau, PHF1 Tau, and MC1 Tau (all phospho-tau antibodies were used at 1:25 and obtained from Dr. Peter Davies, Feinstein Institutes for Medical Research); Tau pS404 (1:1,000, ab92676, AbCam, Cambridge, MA, United States); Tau pS396 (1:1,000, ab92676, AbCam, Cambridge, MA, United States); Tau pT231 (1:1,000, ab151559, Abcam, Cambridge, MA, United States); Tau pS199 (1:1,000, ab4749, AbCam, Cambridge, MA, United States); HSP60 (1:1000, #12165, Cell Signaling Technology, Danvers, MA, United States); Total OxPhos Rodent Antibody Cocktail (1:2,000, ab110413, Abcam, Cambridge, MA, United States); VDAC1 (1:2,000, #4661, Cell Signaling Technology, Danvers, MA, United States); SDHA (1:2,000, #11998, Cell Signaling Technology, Danvers, MA, United States); goat anti-mouse 680RD (P/N: 926-68070), goat anti-mouse 800CW (P/N: 926-32210), donkey anti-rabbit 680RD (P/N: 926-68073) and goat anti-rabbit 800CW (P/N: 926-32211) (1:20,000, Licor, Lincoln, NE, United States).