Figure 6

PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro. (A) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was subjected to Western blot analysis with the PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610) antibodies. When indicated, the PARP inhibitor PJ34 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). (B) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD⁺/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.