The reaction was initiated by addition of the enzyme, and at 0, 5, 10, and 15 min intervals, 10 μl reaction mixture was withdrawn and spotted onto the DE81 filter paper and dried. The unreacted substrate was washed and the products were eluted and counted in a liquid scintillation counter. With [3H]-Gua Doramapimod as substrate

the reaction (in a total of 25 μl) was initiated by addition of the enzyme (10 μl), incubated at 37°C for 2 min, stopped by addition of 1 M HCl (10 μl), and placed immediately on ice. After neutralization, 15 μl of the mixture was spotted onto the DE81-filter paper. The filters were then washed, and the products were eluted and counted by liquid scintillation. IC50 values for purine analogs were determined for both Mpn HPRT and human HPRT using fixed concentrations of [3H]-Hx (10 μM) or [3H]-Gua (10 μM) and variable concentrations of the inhibitors.

Thymidine kinase assay was performed using tritium labelled thymidine ([3H]-dT) as substrate and various concentrations of the inhibitors essentially as previously described [40] to determine the IC50 values of TFT and 5FdU. Kinetic parameters for TFT were determined by using the phosphoryl transfer assays as previously described [52]. Briefly, each reaction was performed in a total volume of 20 μl containing 50 mM Tris/HCl, pH 7.5, 0.5 mg/ml BSA, 5 mM DTT, 2 mM MgCl2, 15 mM NaF, variable concentrations of TFT, 0.1 mM [γ-32P]-ATP, and 50 ng purified enzyme at 37°C for 20 min, and stopped by heating at 70°C for 2 min. After brief centrifugation, 1 μl supernatant was spotted onto a TLC plate KPT 330 (PEI-cellulose, Merck) and dried. The TLC plates were developed in isobutyric acid/ammonia/H2O (66:1:33). The reaction products were visualized and quantified by phosphoimaging analysis (Quantity One, Bio-Rad). Statistical analysis The data were analysed by unpaired student’s t-test (two tailed) using GraphPad Prism 5 software. P < 0.05 is considered as significant. Acknowledgements This work was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and

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