Apyrase from potatoes

The Kinase-Glo Plus Luminescent Kinase Assay kit (Promega, United States) was used to detect the ATPase activity of PEDV nsp13. In brief, the purified recombinant proteins in reaction buffer (40 mM Tris-HCl at pH 7.5, 50 mM NaCl, 2 mM Mg²⁺, 80 μM ATP, and 0.2 μM NTPase) were added to a 96-well black plate in the presence of compounds or DMSO to a total volume of 50 μl. Apyrase from potatoes (Sigma-Aldrich, United States) was used as a positive control enzyme. PEDV N protein was used as a negative control enzyme. The plate was then incubated in a 37°C incubator for 20 min. At the end of the reaction, 50 μl of Kinase-Glo reagent was added to the reaction mixture. After mixing and incubating at room temperature for 2 min, luminescence of each well was measured using a PerkinElmer EnSpire Multimode Plate Reader.

Fetal bovine serum (FBS) was purchased from different commercial sources (Sigma-Aldrich, St. Louis, MO, USA, cat#F7524; Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cat#26140079; Euroclone, Milan, Italy, cat#ECS0180L; Microgem, Naples, Italy, cat # RM10432). Bovine serum albumin (BSA, cat # A2153), apyrase from potatoes (cat # A6535), β-nicotinamide adenine dinucleotide⁺ (NAD⁺, cat # N7004), and hexokinase and glucose-6-phosphate dehydrogenase (cat # H8629) were purchased from Sigma-Aldrich.

Calcium chloride dihydrate was purchased from G-Biosciences (St Louis, MO, USA). Sodium carbonate decahydrate was bought from Alfa Aesar (Lancashire, UK). Magnesium chloride hexahydrate, adenosine 5’-triphosphate (ATP) disodium salt hydrate, Apyrase from potatoes, Dulbecco’s phosphate buffered saline (PBS) with calcium chloride and magnesium chloride was ordered from Sigma-Aldrich Company Ltd. Ammonium phosphate dibasic was obtained from Fluka (Germany). Optimal cutting temperature (OCT) compound was supplied by VWR Chemicals (Leuven, Belgium). All of the chemicals were used without further purification.