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. 2014 Apr 10;9(4):e94149. doi: 10.1371/journal.pone.0094149

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© 2014 Luch et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Flow diagram illustrating how chromatin was dissected to monitor the binding of UV-DDB. Unbound proteins were released by salt (0.3 M NaCl) extraction and the remaining chromatin was solubilized by MNase digestion. (B) Western blot visualization of the chromatin partitioning of UV-DDB using antibodies against DDB2. GAPDH (glyceraldehyde 3-phosphate dehydrogenase), marker of unbound proteins; histone H3, marker of chromatin. Human fibroblasts were exposed for 18 h to formaldehyde or UV-irradiated at the indicated doses. (C) Quantification of three independent binding assays demonstrating the differential interaction of DDB2 with formaldehyde- and UV-damaged chromatin (error bars, S.D.). (D) Release of chromatin-bound DDB2 and histone H3 by high-salt extraction. After incubation with 0.3 M NaCl buffer, the chromatin was dissolved with 2.5 M NaCl, thus liberating non-covalently bound chromatin proteins.