Deae filtermat

Methyltransferase assays were performed in 40 mM Tris-HCl (pH 8.0), 5 mM DTT, 2 μM ^7MeGpppA or GpppA RNA cap analogue (New England Biolabs), 10 μM adenosyl-methionine (AdoMet; Thermo Fisher), 0.03 μCi/μl [³H]AdoMet (PerkinElmer) (25 (link)). For each reaction, MERS-CoV or SARS-CoV nsp14 was added to a final concentration of 500 or 250 nM, respectively. Reaction mixtures were incubated at 30°C for up to 120 min, and reactions were stopped by the addition of a 10-fold volume of 100 μM ice-cold adenosyl-homocysteine (AdoHcy; Thermo Fisher). Then, samples were spotted on a DEAE filter mat (PerkinElmer) prewet with Tris-HCl (pH 8.0) buffer. Filter mats were washed twice with 10 mM ammonium formate (Sigma-Aldrich) (pH 8.0), twice with MilliQ water, and once with absolute ethanol (Sigma-Aldrich). After air drying for 10 min, filter mats were cut, and relevant pieces were transferred to individual tubes. Betaplate scintillation fluid (PerkinElmer) was added, and the amount of ³H label bound was measured in counts per minute using a Wallac scintillation counter. For relative quantification, incorporation measurements for mutant proteins were normalized to values obtained with the wt control nsp14. Samples were measured in duplicate in each experiment.

The TK1 activity in serum samples was determined using [³H]-dThd (PerkinElmer) as the substrate essentially as described [23 (link)]. Briefly, the reaction mixture containing 10 mM Tris–HCl pH 7.6, 2 mM DTT, 5 mM MgCl₂, 5 mM NaF, 5 mM ATP, 5 μM [³H]-dThd, 10 mM NH₄Cl and an appropriate amount of serum in a total volume of 40 μL was incubated at 37 °C for 60 min. Aliquots of the reaction mixture were spotted onto DEAE filter paper (DEAE filtermat, PerkinElmer) and dried. The filters were then washed 2 times in 1 mM ammonium formate. Thereafter, the filters were sorted, eluted in 0.5 ml buffer (0.1 M HCl and 0.2 M KCl) and counted in a scintillation counter (Tri-carb, PerkinElmer) after the addition of scintillation fluid (OptiSafe, PerkinElmer). All samples were assayed at least 3 times, and the results are presented as the mean ± SD.

TK1 activity in serum samples was determined by using [³H]-dThd (PerkinElmer) as the substrate essentially as previously described [26 (link)]. The reaction mixture, containing 10 mM Tris–HCl pH 7.6, 2 mM DTT, 5 mM MgCl₂, 5 mM NaF, 5 mM ATP, 5 μM [³H]-dThd, 10 mM NH₄Cl and 10 μl serum without dilution in a total volume of 40 μL, was incubated at 37 °C for 60 min. Aliquots of the reaction mixture were spotted onto DEAE filter paper (DEAE filtermat, PerkinElmer) and dried. The filters were then washed 2 times in 1 mM ammonium formate. Thereafter, the filters were sorted into vials, the products were eluted with 0.5 ml buffer (0.1 M HCl and 0.2 M KCl) and counted in a scintillation counter (Tri-Carb, PerkinElmer) after the addition of scintillation fluid (Optisafe, PerkinElmer). All samples were assayed at least 3 times and the results are given as mean ± SD.