Figure 1: H-Y-E motif is not the sole indicator for PAR synthesis activity.

GFP–PARPs were immunoprecipitated from 293F cells and subjected to NAD⁺ incorporation reactions in vitro with 5 or 10 μM cold NAD⁺ supplemented with a constant ratio of ³²P-NAD⁺. Automodifed PARPs were resolved on SDS–PAGE gels and subjected to autoradiography. Representative Coomassie stained gels for each PARP purification are shown to the left of the autoradiogram and the expected molecular weight of the PARP indicated by an asterisk. Assays were repeated at least twice. Of the H-Y-E motif containing PARPs, PARP1, 2, 5a and 5b generated polymer, as evidenced by the smear of signal starting from the molecular weight of the PARP. The remaining PARPs resolve as a discreet band, indicating that they do not generate poly(ADPr). See also Supplementary Figs 1–3.