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Immuno-precipitation (IP) and Chromatin IP (ChIP)

Tissue or cell lysates (1 mg/ml protein) in IP incubation buffer (Active Motif, Carlsbad CA), were incubated overnight at 4°C with 20 ?g of antibodies against POLG, PARP1, or PAR molecules. Samples were then loaded on to Protein G Agarose Prepacked Columns (Active Motif), and columns were washed five times with IP wash buffer (Active Motif), to remove non-binding proteins. Immune complexes were eluted from the columns in 0.2 M glycine (pH 2.5) solution. The eluents were pooled (total 100 ?l), neutralized with 20 ?l of 1 M Tris-HCl (pH 9.0), and used for Western blotting.

For ChIP assay, Imprint Chromatin Immunoprecipitation Kit was employed (Sigma). Briefly, Hela cells (5 X 107) or tissue sections (10 mg) were incubated with 9 ml of 1% buffered formaldehyde for 30 min at room temperature on a rocking platform to cross-link DNA/proteins, neutralized by adding 1 ml of 1.25 M glycine buffer, homogenized, and then sonicated on a Misonix XL2020 sonicator (30 pulses, 30 sec on/off, 100% power) to fragment the chromatin DNA. Cross-linked samples were subjected to immune-precipitation with mouse hPARP1 N-terminus-specific monoclonal antibody (sc-74470, Santa Cruz). Mouse IgG (Sigma) was used as negative control. The formaldehyde cross-link was reversed by incubating for 15 min at 65°C. Samples were then treated with RNase H and protease K, to digest RNA and proteins, respectively, and DNA was extracted with phenol/chloroform and purified by using QIAquick PCR Purification Kit (28104, Qiagen). ChIP DNA was used as substrate for PCR amplification of PARP1-bound sequences by using the oligonucleotides listed in S1 Table.

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3:44pm
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