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. 2012 Oct 15;199(2):235–249. doi: 10.1083/jcb.201112132

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© 2012 Pines et al.

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Figure 6. — DDB2 is a target for PARP1-mediated PARylation. (A) The N terminus of DDB2 is targeted for PARylation. In vitro PARylation experiments using purified components reveal that both DDB2 and DDB1 are directly modified by PARP1. Human DDB2 lacking its first 40 N-terminal amino acids including 7 lysines (ΔUV-DDB), failed to undergo PARylation. The zebrafish orthologue of DDB2 (drDDB) lacking the first 93 N-terminal residues is also not PARylated in vitro. (B) 6His StrepII-tag DDB2 isolation using tandem purifications under denaturing conditions. NHF cells stably expressing 6His StrepII-tag DDB2 were irradiated with 100 J/m² UV-C light in the presence or absence of 10 µM PARPi or mock irradiated and incubated for 30 min. The final Strep-Tactin column purifications were separated on SDS-PAGE gels, and proteins were visualized with antibodies against DDB2, PAR, or Ubiquitin. WB, Western blot.