Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)

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	-Mouse (1:1000)

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-Mouse (1:1000)

Publication protocol

For sarkosyl-insoluble fractions, total protein concentration was normalized according to pellet weights. Total protein concentrations for all other samples were determined by Pierce™ Bicinchoninic Acid protein assay (Thermo Scientific). Equal amounts of protein for each sample were loaded and separated using SDS-PAGE on 10%, 10.5–14%, or 10–20% tris-HCl gels (Bio Rad). Protein was transferred to nitrocellulose membranes (Bio Rad), which were blocked with 5% bovine serum albumin (Sigma) in 1× tris buffered saline buffer with Tween 20 (TBST) buffer (10 mM Tris-Base (Sigma), 0.2 M NaCl (Macron Chemicals), 0.1% Tween-20 (Sigma) pH 7.4). Protein was immunoblotted with Tau46 (Cell Signaling Technology #4019, dilution 1:10,000), Tau13 (BioLegend #MMS-520R, dilution 1:60,000), GAPDH(14C10) (Cell Signaling Technology #2118, dilution 1:4000), GAPDH(GA1R) (Thermo Scientific #MA5-15738, dilution 1:5000), AT8 (Thermo Scientific #MN1020, dilution 1:1000), anti-human tau (Abcam # ab74391, dilution 1:10,000), and βIII-tubulin (ProSci #79–720, dilution 1:10,000) antibodies. Additional antibodies from Peter Davies for phospho-epitopes on tau included MC1 (dilution 1:800), CP13 (dilution 1:1,000), and PHF1 (dilution 1:1,500). To visualize antibody immunoreactivity using a LiCor imaging system and Image Studio software, IRDye-linked goat anti-mouse 800CW and goat anti-rabbit 680LT secondary antibodies were used (LI-COR Biosciences,dilution 1:100,000). Following LiCor image acquisition using Image Studio software (Odyssey), Amido black staining solution (Sigma-Aldrich) was used for total protein quantification according to manufacturer’s instructions. Immunoreactivity and Amido black staining were quantified by densitometry using OptiQuant version 3 software, following guidelines for total protein quantification.

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