ENPP1 enzyme activity assay

Evaluation of the ENPP1 activity was carried out with p-Nph-5’-TMP or ATP as the substrate. Enzymatic reactions were performed at 37 °C in a total volume of 100 μL in a clear 96-well plate. The reaction mixture (90 μL) contained 50 mmol/L Tris-HCl (pH 8.5), 130 mmol/L NaCl, 1 mmol/L CaCl₂, 5 mmol/L KCl, 10 μL ENPP1 cell lysate and different concentration of MTX. The enzyme reactions were initiated by the addition of 10 μL of 1 mmol/L p-Nph-5’-TMP dissolved in deionized water. Changes in absorbance due to released p-nitrophenolate were measured at 405 nm every minute for 60 min at 37 °C using a Tecan Spark microplate reader (Tecan, Mannedorf, Switzerland). In the assays where ATP was used as the substrate, the reaction was stopped after 30 min by heating samples at 95 °C for 3 min. The ATP consumption was analyzed by LC-MS/MS (Sciex API-4000). This experiment was performed three independent times.