Fig. 1

NAT10 is a novel substrate of PARylation in response to DNA damage. A, B MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h or 6 Gy IR. Cells were harvested for IP and immunoblotting analyses with the indicated antibodies. C MCF-7 and BT549 cells were treated with or without 1 mM MMS for 2 h and subjected to IP analysis with an anti-NAT10 antibody. A total of 1% SDS was added to lysis buffer to remove all non-covalent binding. The immunoprecipitate was spotted onto a nitrocellulose membrane and the membrane was then examined using an anti-PAR antibody. Immunoblotting analysis was performed with anti-NAT10, PAR, and vinculin antibodies. D In vitro biotin pull-down assays were carried out by incubating purified GST-NAT10 or GST-CHFR with purified PAR (biotin-PAR polymer). GST-CHFR was used as a positive control