Western Blotting of Sarkosyl-Insoluble Fractions

Samples were mixed with loading sample buffer and heated at 95°C for 5 min. Sixty microgram of protein was separated by electrophoresis in SDS-PAGE gels and transferred to nitrocellulose membranes (200 mA per membrane, 90 min). The membranes were blocked for 1 h at room temperature with 5% non-fat milk in TBS containing 0.2% tween and were then incubated with the primary antibody, anti-tau Ser422 (diluted 1:1,000; Thermo Fisher (Waltham, MA, USA). After washing with TBS-T, blots were incubated with the appropriate secondary antibody (anti-rabbit IgG conjugated with horseradish peroxidase diluted at 1:2,000, DAKO, DE) for 45 min at room temperature. Immune complexes were revealed by incubating the membranes with chemiluminescence reagent (Amersham, GE Healthcare, Buckinghamshire, UK) (Ferrer et al., 2018).