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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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Trump confirms June 12 meeting with Kim in Singapore

Kim Yong Chol, the most senior North Korean to visit the United States in 18 years, spent nearly 90 minutes in the Oval Office. But Trump said he didn't like that Kim Jong Un had met Thursday in Pyongyang with Russian Foreign Minister Sergey Lavrov.
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