Fig. 3

NADP⁺ suppresses poly(ADP-ribose) polymerase (PARP) activity in vitro. a PARPs can bind to NADP⁺. HIS-PARP1, HIS-PARP2, and GST-PARP10 were immobilized on Ni Sepharose and glutathione S-transferase (GST) beads respectively, followed by incubating with [³²P]NADP⁺. [³²P]NADP⁺ was heat-released from Ni Sepharose or GST beads and examined by thin-layer chromatography. As the negative controls (NC), Ni Sepharose or GST beads without recombinant PARP proteins was incubated with [³²P]NADP⁺. b, c NADP⁺ suppresses PARP1’s activity in vitro. In vitro PAPR1-mediated PARylation assay was performed using different ratio of NADP⁺/NAD⁺. Auto-PARylation of PARP1 was examined by western blotting (b). [³²P]NAD⁺ was used as the donor in PARP1-mediated in vitro PARylation assay. The auto-PARylation of PARP1 was examined by autoradiography. Coomassie staining of His-PARP1 was shown as the loading control (c). d NADP⁺ suppresses PARP2 in vitro. Auto-PARylation of PARP2 was examined by western blotting with the indicated antibody. e NADP⁺ suppresses PARP10 in vitro. Auto-MARylation level of PARP10 was examined by autoradiography