Adenosine triphosphate (atp)

The ADP-Glo kinase assay (Promega, Madison, WI, USA) was used to screen CHMFL-KIT-031 for its c-KIT and the relevant mutations inhibition effects. The kinase reaction system contains 9 μL c-KIT (20 ng/μL) or c-KIT V559D (15 ng/μL), 1 μL of serially diluted CHMFL-KIT-031, and 10 μL substrate Poly (4:1 Glu, Tyr) peptide (0.4 μg/μL) (Promega, Madison, WI) with 100 μM ATP (Promega, Madison, WI). The reaction in each tube was started immediately by adding ATP and kept going for an hour under 37°C. After the tube cooled for 5 minutes at room temperature, 5 μL solvent reactions were carried out in a 384-well plate. Then 5 μL of ADP-Glo reagent was added into each well to stop the reaction and consume the remaining ATP within 40 minutes. At the end, 10 μL of kinase detection reagent was added into the well and incubated for 30 minutes to produce a luminescence signal. Luminescence signal was measured with an automated plate reader (Envision, PE, USA) and the dose-response curve was fitted using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The biochemical tests of other targets were provided by Invitrogen (Carlsbad, CA, USA).

One million Mo-MDSCs purified from bone marrow, spleen and tumour were lysed by cell lysis buffer (Cell Signaling Technology), and immunoprecipitated by anti-CDK2, and Protein A Dynabeads (Thermo Fisher Scientific). The antibodies are listed in Supplementary Data 4. For kinase reaction, precipitated CDK2 was incubated in 20 mM Tris-HCl (pH7.5) containing 5 ng/µl of recombinant SMAD3 (Sigma-Aldrich), 100 µM ATP (Promega), 0.1 mg/ml BSA, 1 mM DTT and 40 mM MgCl₂ at 30 °C for 2 h. ATP to ADP conversion during kinase reaction was measured by ADP-Glo Kinase Assay (Promega) and Powerscan HT (DS Pharma Biomedical).

shFBP1-RD stable cells were grown to 90% confluence in DMEM. Cells were washed and scraped with PBS, and then pelleted by centrifugation at 300 × g for 10 minutes at 4°C. After discarding the supernatants, the cell pellets were resuspended in 1.5x pellet volume of hypotonic lysis buffer (10 mM HEPES-KOH, pH 7.6, 10 mM KOAc, 0.5 mM Mg(OAc)₂, 2 mM DTT, and 1x protease inhibitor cocktail [Roche]), placed on ice for 30 minutes, and then homogenized with a 27-gauge 1/2-inch needle. Cell extracts were centrifuged at 10,000 × g for 20 minutes at 4°C, and the supernatants were recovered and stored at −80°C. In vitro IRES activity assays were performed in a final volume of 25 μl containing 0.25 μg EV71 5′ UTR-Luc reporter RNA, 60% volume of RD shFBP1 cell extracts, 10 mM creatine phosphate, 50 μg/ml creatine phosphokinase, 79 mM KOAc, 0.5 mM Mg(OAc)₂, 2 mM DTT, 0.02 mM hemin, 0.5 mM spermidine, 20 mM HEPES-KOH (pH 7.6), 20 μM amino acid mixture (Promega), 0.4 mM ATP (Promega), and RNase inhibitor. The reactants were incubated at 30°C for 90 minutes, and firefly luciferase activity was measured using the luciferase assay system (Promega).

Cell extract representing the soluble protein fraction prepared from seedlings in luciferase assay buffer [20 mM Tricine, pH 7.8, 1.07 mM (MgCO₃)₄Mg(OH)₂-5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM CoA, and 500 μM ATP (Promega)], and the reaction was initiated with the injection of 100 μL of 0.5 mM luciferin in luciferase assay buffer. Photons were counted using a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). Each mRNA construct was assayed in duplicate and the average is reported. Protein concentration was determined as described [94 (link)].

The reactions were carried out in a total volume of 25 μL in 96-well microtiter plates. The Syk tyrosine kinase activity at single dose concentration of 12.5 ng/μL, 10 μL of volume, was carried out served as the enzyme source. The total volume of 10 μL mixture containing 0.2 μg/μL Poly (Glu, Tyr) sodium salt (4:1, Glu:Tyr, Sigma–Aldrich, St. Louis, MO, USA) and 10 μM ATP (Promega, Madison, WI, USA) served as the standardized substrate. The concentration range of the tested inhibitors employed in reactions was 0.0032, 0.016, 0.08, 0.4, 2, 10 μM or DMSO with 5 μL volume. All of the enzymatic reactions were conducted at 37 °C for 60 min. The assay was terminated by adding 25 μL of ADP-GloTM Reagent (Promega, Madison, WI, USA). The 96-well plate was shaken and then incubated for 40 min at ambient temperature. Fifty microliter of Kinase detection reagent was added and the 96-well reaction plate was then read using the ADP-Glo Luminescences Protocol on a GloMax plate reader (Promega: Catalog #E7031). For each concentration of Vam3, the rate of reaction at each concentration of ATP was determined and plotted against the ATP concentration to determine the apparent K_m and V_max (maximal rate). Finally, the apparent K_m (or apparent K_m/V_max) was plotted against the inhibitor concentration to determine the K_i. All data analysis was performed using Prism and Prism enzyme kinetics programs.