Samples were denatured by boiling in Laemmli buffer for 5 min. Protein concentration was measured using the Pierce™ 660 nm Protein Assay Kit (ThermoFisher Scientific). Samples were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Millipore Sigma, Burlington, MA, USA). The membrane was subsequently blocked with 5% fat-free milk-TBS (Tris-buffered saline) for 30 min, incubated with primary antibodies (Table (Table1)1) in 5% fat-free milk-TBS overnight, washed with TBST (TBS containing 0.05% Tween 20), incubated with peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), washed with TBST, incubated with the ECL Western Blotting Substrate (ThermoFisher Scientific) and exposed to HyBlot CL® autoradiography film (Denville Scientific Inc., Holliston, MA, USA). Specific immunosignal was quantified by using the Multi Gauge software V3.0 from Fuji Film (Minato, Tokyo, Japan).

Antibodies used in this study

Mono- monoclonal, p- phosphorylated, up- unphosphorylated, Poly- polyclonal, M mouse, R rabbit

Tau level in samples was assayed by immuno-dot blots as described previously. Briefly, various amounts of a sample were applied onto nitrocellulose (NC) membrane (Schleicher and Schuell, Keene, NH, USA) at 5 μl per grid of 7 × 7 mm in size. The blot was placed in a 37 °C oven for 1 h to allow the protein to bind to the membrane. Then, the membrane was processed as for Western blots described above by using a mixture of R134d and 92e pan tau antibodies as primary antibodies.

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