3.2.5. Inhibitors Screening, ADP-ribosylation of DNA by PARP-3

The reactions were performed in the presence of 2 mM MgCl<sup>2</sup> as a cofactor in HDB buffer solution containing HEPES-KOH, pH 8.6, 0.25 mg/mL BSA and 0.5 mM DTT. The reaction mixtures (final volume 10 µL) contained 0.02 µM [32P]-DNA substrate, 0.1 µM PARP-3, 5, 10, 100 or 500 µM NAD+ in the absence or presence of various concentrations of the inhibitor, which are indicated on the figures. The reaction was carried out for 20 min at 37 ◦C. The mixtures were separated in a standard 20% denaturing acrylamide gel. The gels were dried and subjected to autoradiography and/or phosphorimaging for quantitation using the Typhoon imaging system (GE Healthcare Life Sciences, Chicago, IL, USA). Quantitative processing was carried out using OriginPro7.5, Microcal Software (Origin Systems, Houston, TX, USA). In all experiments, the points on the experimental curves represent the average of minimum three independent experiments. Standard deviation did not exceed 10%.
