Kinase glo kit

HUVECs or retinae were lysed in RIPA buffer and immediately subjected to the NDP kinase activity assay. The test utilizes the transphosphorylase activity of NDPK to generate ATP from GTP and ADP. The newly-formed ATP is directly used by firefly luciferase to convert D-luciferin to the light-emitting oxyluciferin with the Kinase-GLo kit (V6711, Promega, Germany). Under the experimental conditions, the luminescent signal is proportional to the amount of ATP produced, and thus to the enzymatic activity of NDPK. A standard curve was generated by adding increasing concentrations of ATP (0.5–10 µM) in the absence of NDPKs. The NDP kinase activity in cell lysates was measured in a 384-well plate. Lysates were diluted in assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 1 mM dithiothreitol (DTT), and 0.01% bovine serum albumin (BSA). The readout was commenced by adding the luciferase substrate (V6711, Kinase-GLo, Promega, Germany). After 5 min of incubation, the luminescence was recorded at room temperature using a multiplate reader (EnVision, PerkinElmer, Germany).

Test compounds were purchased from commercial sources. Caffeic acid and rosmarinic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Salvianolic acids A and B (Sal A and Sal B) were purchased from TAUTO (Shanghai Tauto Biotech Co. Ltd., Shanghai, China) and Sal C was purchased from Herbest (Baoji Herbest Bio-Tech Co. Ltd., Baoji, China). All the tested compounds were of HPLC grade with ≥98% purity. Human recombinant GSK-3β was obtained from Prospec (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel). GSM substrate mimicking glycogen muscle synthase was obtained from Merck Millipore (Millipore Corporation, Temecula, CA, USA) and a Kinase-Glo kit was obtained from Promega (Promega Corporation, Madison, WI, USA). Adenosine 5-triphosphate (ATP) disodium salt hydrate, ammonium hydroxide, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), ammonium acetate, ethylene glycol-bis(-aminoethylether)-N,N,N′,N′-tetraacetic acid tetrasodium salt (EGTA), formic acid, ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione, and magnesium acetate tetrahydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were purchased from E. Merck, Fluka (Rupert-Mayer-Str., Munich, Germany) and Sigma-Aldrich unless otherwise stated.

His-PRPS1 and His-PRPS2 were expressed in E.coli BL21 cells and purified using a Ni²⁺–Sepharose affinity column (cytiva, 17-5248-01). NAD/NADH/GDP/ADP were diluted to the corresponding concentration with assay buffer (50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM Na₃PO₄, 1 mM DTT, 1 mM EDTA, 600 µM ATP, 600 µM R-5-P). Enzyme (50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM Na₃PO₄, 1 mM DTT, 1 mM EDTA, 2 μg/mL PRPS1 or 2) was blended with the mixture in equal volume for a final reaction system (50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM Na₃PO₄, 1 mM DTT, 1 mM EDTA, 300 µM ATP, 300 µM R-5-P, 1 μg/mL PRPS1 or 2). The reaction was incubated at 37°C for 10 min and terminated by adding an equal volume of 10 mM CaCl₂. For the kinetics study, the activity of PRPS1/2 was measured based on ATP consumption that was detected according to the manufacturer’s instructions of the Kinase-Glo Kit (Promega, V6072).

The half maximal inhibitory concentration (IC₅₀) values of Lycorine and positive control Gefitinib against EGFR kinase activity were carried out using the Promega Kinase-Glo kit (Promega, Mannheim, Germany) according to the manufacturer’s protocol in the presence of 600 nM ATP. Data were presented as means and 95% confidence intervals (CIs) from three independent experiments.