3.2.6. Inhibitors Screening, ADP-ribosylation of PARP-3

The reactions were performed in the presence of 2 mM MgCl<sup>2</sup> as a cofactor in HDB buffer. The reaction mixtures (final volume 10 µL) contained 0.1 µM DNA substrate, 0.5 µM PARP-3, 5 µM [ <sup>32</sup>P]-NAD+ and 0, 35 or 95 µM NAD+ in the absence or presence of various concentrations of the inhibitor, which are indicated on the figures. The reaction mixtures were incubated for 20 min at 37 ◦C then terminated by adding the 5x gel loading Laemmli sample buffer and heating at 85 ◦C for 10 min. The mixtures were resolved on a 10% SDS-PAG as described in [84] The gels were dried and subjected to autoradiography and/or phosphorimaging for quantitation using the Typhoon imaging system from GE Healthcare Life Sciences. Quantitative processing was carried out using OriginPro7.5, Microcal Software, USA. In all experiments, the points on the experimental curves represent the average of a minimum three independent experiments. Standard deviation did not exceed 10%.
