Tau151-391 tagged with hemagglutinin (HA) was overexpressed in HEK-293FT cells. The cells, 48 h after transfection, were lysed in phosphate-buffered saline (PBS) containing 50 mM NaF, 1 mM Na3VO4, 1 mM AEBSF, 5 mM benzamidine, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin by probe sonication at 20% power for 2 min. The cell lysates were centrifuged for 10 min at 10,000×g. The extract containing HA-tau151-391 was stored at − 80 °C until use.

Various amounts of AD tau fractions were dotted on nitrocellulose membrane and dried at 37 °C for 1 h. The membrane was blocked with 5% fat-free milk-TBS for 1 h and incubated with the above cell extract containing HA-tau151-391 overnight. After washing with TBST, captured HA-tau151-391 was detected by incubating with anti-HA in 5% milk-TBST and processed as described above for immuno-dot blots.

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