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For protein extraction, cells were collected by scraping in cold PBS, washed with cold PBS, and then lysed in RIPA buffer (Wako, Japan) containing a protease inhibitor cocktail (Sigma, P8340) for 20 min on ice; the resulting lysate was cleared by centrifugation at 10,000×g and 4 °C for 10 min. The protein content was quantified by a BCA assay (Thermo Scientific), and equal amounts of protein from each sample were separated by SDS–PAGE (Wako, Japan) and transferred to a PVDF membrane (Bio–Rad). GAPDH was used as the loading control for normalisation. Bands were visualised using ImageQuant LAS4000 (GE technology). Images of western blots were obtained using ImageJ.

200–500 mg samples of frozen frontal and temporal cortex were dissected from selected tau-immunopositive cases (see later) and subjected to western blot analysis of insoluble tau, as we have described elsewhere [32 ]. Briefly, sarkosyl-insoluble pellets were prepared by homogenization of tissue samples in 20vol (v/w) of extraction buffer containing 10 mM Tris–HCl (pH 7.5), 0.8 M NaCl, 10 % sucrose, 1 mM EGTA, 2 % sarkosyl and incubated for 30 min at 37 °C. After centrifugation at 20,000 g for 10 min at 25 °C, the supernatants were taken, transferred to 1.5 mL tubes and ultracentrifuged at 100,000 g for 20 min at 25 °C. The pellets were washed by ultracentrifugation with 0.5 mL of sterile saline, solubilized in SDS-sample buffer and subjected to 4–20 % gradient polyacrylamide gel (Wako) SDSPAGE. Proteins were transferred to PVDF membrane, incubated overnight with the anti-tau monoclonal antibody T46 (Thermo Scientific), biotinylated 2nd antibody, avidin–biotin complex (Vector) and developed with diaminobenzidine and nickel chloride.

Cells (5 × 10⁶) were washed once with PBS (without calcium) and sonicated in cold buffer (20 mM Tris, 2.5 mM Sodium Pyrophosphate, 150 mM NaCl, 1 mM Disodium β-Glycerophosphate Pentahydrate, 1 mM ethylenediaminetetraacetic acid, 1 mM 14-bis[(acetyloxy)methyl] ester, 1% Triton-X) for 5 min. The lysates were clarified by brief centrifugation (10,000 × g), and the supernatant was used for analysis. After measuring protein concentrations with a protein assay kit (Bio Rad, Richmond, CA), 20 μg protein of each sample was mixed with an equal volume of SDS-loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% β-mercaptoethanol), boiled for 5 min, and subjected to SDS-PAGE (Wako, Japan). The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA) that was incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a West Pico chemiluminescent substrate kit (Pierce, Rockford, IL), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan).