*3.1. Tau Expression and Purification*

Wild-type (wt) Tau (2N4R isoform; 441 residues) plasmid (Addgene plasmid #16316, a gift from Peter Klein) was transformed into *Escherichia coli* BL21 star cells. Cells were grown at 37 ◦C in Terrific Broth medium in the presence of kanamycin until the optical density at 600 nm (OD600) reaches 0.8–1.0, then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown overnight at 18 ◦C.

wt Tau was purified using a similar procedure described by Barghorn et al. [43]. Briefly, wt Tau cell pellets were resuspended in 50 mM NaCl, 5 mM DTT, 50 mM sodium phosphate, pH 6.5, and supplemented with a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA). The cells were lysed using a homogenizer (Avestin, Ottawa, ON, Canada). Additional salt was then added (for a final concentration of 450 mM NaCl) before the solution was incubated for 20 min in hot water (~80–90 ◦C). The supernatant was concentrated, diluted to a final salt concentration of 50 mM NaCl, and purified by FPLC (Bio-Rad, Hercules, CA, USA) using a salt gradient applied to a heparin sepharose HP column (GE, Marlborough, MA, USA). Fractions containing wt Tau were concentrated and further purified by reverse-phase HPLC (Agilent, Santa Clara, CA, USA), lyophilized, and stored at −80 ◦C until later use. Purified acetylated Tau (Ac-Tau) was prepared using reverse-phase HPLC after in vitro acetylation of wt Tau (see below).
