Kinasemax kit

Human tRNA^Tyr transcript (HGNC symbol: TRY-GTA2–1) was transcribed overnight at 37°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol. The reaction was gel purified using a Novex TBE-Urea gel (Fisher Scientific) followed by ethanol precipitation. The purified transcript was labeled at the 5′-end with [γ−³²P]ATP using the KinaseMax kit (Fisher Scientific) and incubated with an increasing concentration gradient of recombinant human TyrRS (WT and K244Q) from 125 nM to 8 μM in 10 mM HEPES, pH 7.5, 4 mM KCl, 0.02 mg/mL BSA, and 0.2 mM DTT for 30 min at RT. Each reaction was repeated in triplicate and samples were applied to nitrocellulose and nylon membranes using a Bio-Dot microfiltration apparatus (Bio-Rad) according to methods described in Rio.^{62 (link)} Membranes were washed two times with 60 mM HEPES, pH 7.5, and 10 mM MgCl₂ to remove the unbound tRNA. Membranes were then exposed to a phosphorscreen overnight which was then scanned with a Typhoon imager (Cytiva). The fraction of TyrRS bound tRNA was quantified from the scan with ImageJ^{54 (link)} and plotted using GraphPad Prism (GraphPad Software).

All DNA and RNA oligonucleotides used in this study were purchased from Integrated DNA Technologies (IDT DNA, Coralville, IA). For 5′-end labelling of oligonucleotides with [γ32 P]ATP, the Ambion KinaseMax kit was used followed by purification through Ambion NucAway spin columns, according to protocols provided by the supplier (Thermo Fisher Scientific Inc, Mississauga, ON).
Sofosbuvir (SOF) and its active triphosphate form (SOF-TP) were kindly provided by Gilead Sciences Inc. Mycophenolic acid (MPA) was obtained from Sigma-Aldrich (Markham, ON).

Freshly isolated untreated CD-1 mouse liver samples were homogenized in cold Dulbecco’s PBS (DPBS) containing protease inhibitor cocktail (Roche) with a glass/Teflon homogenizer. Homogenates were centrifuged at 4°C for 20 min at 21 000× g to remove insoluble cellular debris. Next, the protein-containing supernatant was transferred to fresh tubes, and protein concentration was determined by the BCA assay (Pierce). Protein concentrations were adjusted to 4 mg/ml with DPBS and glycerol at 1:0.6 (lysate:glycerol) ratio. Samples were aliquoted into single dispense tubes and then stored at −80°C. LNA-modified ASOs were 5′-end labeled with [γ ³²P]ATP (6000 Ci/mmol, 10 mCi/mL, Perkin Elmer) and T4 polynucleotide kinase with the KinaseMax™ kit (Ambion) according the supplied protocol with one modification. Binding reactions were set up in 20 µl reactions by mixing of 30 000 cpm (∼1–5 pmol) of LNA compounds with 40 µg of prepared liver lysate. The reactions were incubated for 30 min at room temperature and subsequently resolved by 6% non-denaturing polyacrylamide gel electrophoresis. Gels were dried using a BioRad gel drier and exposed on phosphor imaging screens overnight. The phosphor screens were scanned and quantified using the Perkin Elmer Cyclone Plus® storage phosphor system and associated software.