In rnase inhibitor

The gel shift assay was performed with recombinant full length 4R2N and K18 tau and chromatographically purified unacetylated yeast tRNA^Lys (tRNA Probes) in 100 mM sodium acetate buffer at pH 7.0. The molar concentration of tRNA^Lys was accurately remeasured with UV spectrophotometry after base-hydrolization to account for the hyperchromic effect from the secondary and tertiary structure of tRNA [36 (link)]. RNA43 was from Pierce RNA 3ʹ end biotinylation kit (Thermo Scientific, Waltham, MA) with the sequence of 5ʹ-CCUGGUUUUUAAGGAGUGUCGCCAGAGUGCCGCGAAUGAAAAA-3ʹ. The hydrolyzed tRNA and RNA43 samples were then quantified with UV spectrophotometry at 260 nm using an extinction coefficient of 0.025 (μg/ml)^-1cm^-1. For the gel shift assay, protein was incubated with tRNA at 37°C for 10 minutes in the presence of 0.5 mM EDTA, 0.5 mM MgCl_2, 2 U SUPERase• In RNase Inhibitor (Thermo Fisher, Waltham, MA), 0.01% IGEPAL CA-630 (Sigma-Aldrich), and then applied to a TBE 8% Polyacrylamide Gel (Thermo Fisher, Waltham, MA). After gel separation, tRNA was stained with SYBR Gold II (Thermo Fisher, Waltham, MA). For quantitative analysis, the fraction of free and bound tRNA was quantified in ImageJ2 (National Institute of Health, Bethesda, MD) [69 ].

Polysome profiles were obtained according to a previously published procedure (Göktaş et al., 2017 (link)). Briefly, cell lysis (3×10⁷ cells) was carried out in 5 mL lysis buffer [(100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 μg/mL cycloheximide (Applichem) and 30 U/mL SUPERase.IN RNase inhibitor (Ambion)], and the lysate was incubated on ice for 8 min. The cell debris and nuclei were removed by centrifuging the homogenates at 12,000 g at 4 °C for 8 min. Two-mL supernatant was loaded onto 5%–70% (w/v) sucrose gradients [100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 200 U SUPERase.IN RNase inhibitor (Ambion)] and centrifuged at 27,000 rpm for 2 h 55 min at 4 °C in a Beckman SW28 rotor. Fractions were collected using Teledyne ISCO’s density gradient fractionation system (NE, USA) while recording the absorbance at A₂₅₄ to obtain the polysome profiles.