1 step nbt bcip substrate

Guide/donor oligonucleotide containing gfp2bfp-sgGFP oligo was cloned into pZS160 (Table S4). The plasmid was transformed into 3 yeast strains: BY4742, ZRS81 and ZRS82 as described above. Yeast transformants were grown in YNB -HIS/-URA 2% raffinose for 18-24 hours and YNB -HIS/-URA 2% galactose for 48 hours with 1:30 dilution every 24 hrs. Cell pellets were homogenized with glass beads in Trizol (Invitrogen) and total RNA was extracted following the manufacturer’s instructions. Total RNA were further treated with RQ1 DNase (Promega) or RNase A (Invitrogen) before ethanol precipitation. Untreated and nuclease-treated total RNA were loaded to Novex 10% Urea-TBE gels (Thermo Fisher Scientific) for size separation and bands were visualized by staining with SYBR GOLD before transferring to Hybond N+ nylon membranes (GE healthcare) for blotting and UV-crosslinking. For Southern blot analysis, 5’- Digoxigenin labelled DNA probe (Integrated DNA Technologies) targeting msDNA (homology to BFP) was hybridized to the membrane at 55C in DIG Easy Hyb buffer (Roche) (Table S4). Membranes were then washed with 2x SSC, 0.5% SDS twice at 55C, hybridized to anti-Digoxigenin-AP antibody (Roche) and washed 3 times in 1x PBS-T. Antibody localization was visualized by addition of 1-Step NBT/BCIP substrate (Thermo Fisher Scientific).

Whole cell extracts were prepared by harvesting cells in lysis buffer A (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% NP-40, 0.1% sodium deoxycholate, 0.01% SDS) supplemented with 1 × HALT protease inhibitor cocktail (ThermoFisher) and 1 × phosphatase inhibitor cocktail 3 (Sigma). Whole cell lysates were cleared from debris by centrifugation at 20,000 × g for 5 minutes at 4°C. The supernatant (about 30 μg protein per lane) was separated under reducing conditions on 8% tris-glycine mini gels (Invitrogen) following the manufacturer’s protocol, blotted onto PVDF membranes, and labeled with antibodies following standard procedures. For a list of primary antibodies, antibody concentrations and blocking conditions see Table 1. All tau phosphorylation sites throughout the manuscript, in Table 1, and in the figures are referred to by their amino acid position in the human 2N4R tau protein. Please see Figure S1A in the supplementary material for the corresponding sites in the mouse protein. Alkaline-phosphatase-conjugated secondary antibodies diluted 1:5,000 in 5% (w/v) dry milk powder in TBST (tris buffered saline with 0.1% Tween20) and 1-step NBT/BCIP substrate (ThermoFisher) were used for protein detection.