incubated on ice for additional 30 minutes. The samples were centrifuged for 1 hour at 100,000 × g and the supernatant fractions were collected as TBS-insoluble/2% SDS-soluble fractions. The 2% SDS-insoluble pellets were briefly washed with SDS extraction buffer and then further extracted with 90% formic acid (Sigma-Aldrich) on ice and centrifuged for 1 hour at 100,000 × g to get TBS-insoluble/2% SDS-insoluble/formic acid-soluble fractions. The formic acid fractions were enriched by using SpeedVac and neutralized by 2 M Tris-Cl buffer (pH 8.3). Protein levels of SDS-soluble fractions were used to normalize the total protein levels in TBS and formic acid fractions. Puri3cation of sarkosyl-insoluble tau was performed as previously described28,29 with modi3cations. TBS-insoluble pellets were resuspended in 1% sarkosyl/RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% w/v sodium deoxycholate, 2% v/v NP-40, 1% w/v N-Lauroylsarcosine, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific), 1 mM NaF, 1 mM NaVO3, 2 mM PNT (EMD Millipore) and 1 mM PMSF (Sigma-Aldrich) and incubated on ice for 1 hour. The samples were then centrifuged for 1 hour at 100,000 × g and the supernatant fractions were collected as 1% sarkosyl/RIPA-soluble fractions. The insoluble
