Fig. 2

PARP1 PARylates NAT10 at K1016, K1017, and K1020 both in vitro and in vivo. A Four GST-NAT10 deletion mutants (∆1–201, ∆202–488, ∆489–753, and ∆754–1025) and GST control were bacterially purified, and subjected to in vitro PARation assay in the presence of PARP1 and NAD⁺. The reaction samples were resolved by SDS-PAGE, and analyzed by immunoblotting analyses with anti-PAR and anti-GST antibodies. B In vitro PARylation assays were performed using purified GST-NAT10 deletion fragments in the presence or absence of recombinant PARP1 enzyme, NAD⁺, and Olaparib. PARylated NAT10 was detected with an anti-PAR antibody. C–E GST-NAT10 deletion fragments were subjected to in vitro PARation assay as described in A. F, G Purified GST-NAT10 754–1025 proteins (WT, K1016A, K1017A, D1018A, and K1020A, K3A) were subjected to in vitro PARation assays in the presence of PARP1 and NAD⁺. PARylation of NAT10 was detected by immunoblotting with an anti-PAR antibody. In G, K3A represents the combined mutation in all three residues (K1016, K1017, and K1020). H Alignment of the NAT10 protein sequence among different organisms. Asterisk (*) indicates the full conservation of the residues of NAT10 among different species. I, J MCF-7 cells were transfected with HA-NAT10 or HA-NAT10 K3A expression vector. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (I) or 6 Gy IR (J). Thereafter, IP and immunoblotting analyses were conducted with the indicated antibodies