3h dthd

STK1 activities in the serum samples were determined using an optimized [³H]-dThd phosphorylation assay as described previously [9 (link)]. In brief, 10 μL of serum was incubated with a reaction buffer containing 20 mM Tris/HCl, pH 7.6, 2 mM dithiothreitol (DTT), 5 mM sodium fluoride (NaF), 5 mM MgCl₂, 5 mM adenosine triphosphate (ATP) and 5 μM [-³H]-dThd (20 Ci/ml, PerkinElmer, Boston, MA, USA). The reaction mixture was incubated at 37°C for 1 h. The radioactivity in the reaction products was determined as described previously [9 (link)]. Thymidine kinase 1 activities were expressed as pmol dTMP formed per min per mL of serum.

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.