Adenosine triphosphate (atp)

Nucleotide analog inhibitors were serially diluted 3-fold in water and added to reaction mixture containing 10% virus cRNP (v/v), 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM DTT, 10% glycerol, 0.25% Triton-101 (reduced), 5 mM MgCl₂, 0.4 U/mL RNasin, and 200 μM ApG dinucleotide primer (TriLink, San Diego, CA). Reactions were initiated by addition of ribonucleotide triphosphate (NTP) substrate mix containing 1 μM GTP (at 0.5 x K_m), 0.01 μM α-³³P GTP, and 100 μM of ATP, CTP, and UTP (PerkinElmer, Shelton, CT). Reactions were incubated at 30°C for 90 minutes then spotted onto DE81 filter paper. Filters were air dried, washed with 0.125 M Na₂HPO4 (3×), water (1×), and Ethanol (1×) and air dried before exposure to Typhoon phosphor imager screen. Product formation was quantified on a Typhoon Trio (GE Healthcare, Piscataway, NJ). IC₅₀ values were calculated for inhibitors by fitting the data to a four-parameter sigmoidal dose response with variable slope in GraphPad Prism.

The in vitro kinase assay was performed using four previously described GST‐tagged FANCA fragments (#1 to #4) 11. Purified GST‐tagged FANCA fragments were incubated with 85 ng of Aurora A kinase protein (Cell Signaling Biotechnology, Danvers, MA, USA) in kinase buffer composed of 25 mm Tris–HCl (pH7.4), 10 mm MgCl₂, 2 mm DTT, 200 μm cold ATP, and 5 μCi [γ‐P³²] ATP (Perkin Elmer, Waltham, MA, USA) at 30 °C for 30 min. Samples were subjected to SDS/PAGE and transferred to nitrocellulose membranes (GE Healthcare, Milwaukee, WI, USA), followed by autoradiography to detect phosphorylated protein.

CCK was from American Peptide (Sunnyvale, CA); Medium 199 was from GIBCO (Grand Island, NY). ATP and [γ-32P] ATP were from Perkin Elmer (Torrance, CA). CRT0066101 and CID755673 were obtained from TOCRIS (Mo, USA). Nitrocellulose membranes were from Schleicher and Schuell BioSience. Carbachol and GF1 (also known as GF109203X or bisindolylmaleimide I) were from Calbiochem (La Jolla, CA). Antibodies against PKD C-20, IκB-α, or LDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphoserine 744/748 PKD antibody that detects primarily the phosphorylated state of Ser 744 (Jacamo et al., 2008 (link)), phosphoserine 916 PKD antibody, antibodies for NF-κB P65, phosphoserine 32/36 IκB-α, GAPDH, ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). IL-6 antibody was from PeproTech (Rocky Hill, NJ) and MCP-1 antibody was from Antibodies-Online Inc. (Secaucus, NJ). Protein-A-agarose was from Roche Applied Science (Mannheim, Germany) and PKD substrate syntide-2, was from Bachem (Chicago, IL). Other items were from standard suppliers or as indicated in text.

All oligonucleotides used for the in vitro biochemical assays were purchased from Keck Biotechnology Resource Laboratory at Yale University and purified by denaturing polyacrylamide gel electrophoresis. [γ-³²P]ATP (5 mCi) and ATP were purchased from PerkinElmer Life Sciences and Sigma, respectively. T4 polynucleotide kinase was purchased from New England Biolabs. 5-fluorouracil was purchased from Sigma. The ATR inhibitor VE-821 was purchased from Selleckchem.

0.33 μM His-RNMT (WT or T77A)-GST-RAM 1–90, 0.01 μmol CDK1-Cyclin B1 (New England Biolabs), 100 μM ATP (cold or [γ-³²P] ATP [500 cpm/pmol]) (PerkinElmer), 10 mM MgCl₂, 2 mM DTT, 50 mM Tris (pH 7.5), and 0.1 mM EGTA were incubated in 30 μl at 30°C while shaking at 1,200 rpm. Following SDS-PAGE, RNMT bands were excised and analyzed by scintillation counter to estimate the stoichiometry of ³²P incorporation into RNMT.
Enzyme kinetics were measured using 0.01 μM CDK1-Cyclin B1 and a titration of His-RNMT GST-RAM 1–90 or histone H1 using the previous conditions. Phosphorylated protein was quantified following SDS-PAGE and autoradiography by image densitometry. Slopes calculated from linear regression obtained in Figure S1 were plotted as the reaction velocities in Figure 3D.