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. 2014 Apr 25;42(10):6337–6351. doi: 10.1093/nar/gku288

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Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Figure 7. — Intrinsic cross-linking of PARP-1 to AP site-containing DNA in vivo. (a) The reaction scheme of covalent cross-linking of PARP-1 in extracts and the schematic representation of the ³²P-labeled uracil-containing DNA substrate are illustrated. (b) Intrinsic cross-linking of PARP-1 to AP site DNA in cell extracts. Mouse fibroblast cell extracts from wild-type (PARP-1^+/+) cells (lanes 1, 4 and 5) and PARP-1 null (PARP-1^−/−) cells (lanes 2 and 3) were incubated with ³²P-5′-end labeled uracil-containing DNA substrate (depicted in the panel a) without NaBH₄. The reaction mixture in lane 3 was supplemented with purified PARP-1 (100 nM). After incubation, the reaction mixtures in lanes 4 and 5 were subjected to immunoprecipitation analysis with non-immune IgG or anti-PARP-1 IgG, as indicated. The cross-linked products were analyzed as in Figure 1. The symbol () represents PARP-1. The migration position of the incised DNA strand adducted to PARP-1 is indicated. (c) Intrinsic covalent cross-linking of PARP-1 to genomic DNA in mouse fibroblasts treated as indicated. DPCs were isolated as described under Materials and Methods. Samples were from control untreated cells (lane 2), 4-AN-treated cells (lane 3), MMS-treated cells (lane 4) or cells treated with the MMS and 4-AN combination (lane 5). Samples were analyzed by SDS-PAGE and immunoblotting with anti-PARP-1 antibody. Lane 1 corresponds to a sample of purified PARP-1 used as a positive control. A representative image of three experiments is shown.

graphic file with name gku288ufig1.jpg — Intrinsic cross-linking of PARP-1 to AP site-containing DNA in vivo. (a) The reaction scheme of covalent cross-linking of PARP-1 in extracts and the schematic representation of the ³²P-labeled uracil-containing DNA substrate are illustrated. (b) Intrinsic cross-linking of PARP-1 to AP site DNA in cell extracts. Mouse fibroblast cell extracts from wild-type (PARP-1^+/+) cells (lanes 1, 4 and 5) and PARP-1 null (PARP-1^−/−) cells (lanes 2 and 3) were incubated with ³²P-5′-end labeled uracil-containing DNA substrate (depicted in the panel a) without NaBH₄. The reaction mixture in lane 3 was supplemented with purified PARP-1 (100 nM). After incubation, the reaction mixtures in lanes 4 and 5 were subjected to immunoprecipitation analysis with non-immune IgG or anti-PARP-1 IgG, as indicated. The cross-linked products were analyzed as in Figure 1. The symbol () represents PARP-1. The migration position of the incised DNA strand adducted to PARP-1 is indicated. (c) Intrinsic covalent cross-linking of PARP-1 to genomic DNA in mouse fibroblasts treated as indicated. DPCs were isolated as described under Materials and Methods. Samples were from control untreated cells (lane 2), 4-AN-treated cells (lane 3), MMS-treated cells (lane 4) or cells treated with the MMS and 4-AN combination (lane 5). Samples were analyzed by SDS-PAGE and immunoblotting with anti-PARP-1 antibody. Lane 1 corresponds to a sample of purified PARP-1 used as a positive control. A representative image of three experiments is shown.