Molecules and Cells

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Fig. 2.

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Fig. 2. (A) Recombinant PARP1 and RNF168 were subjected into in vitro ubiquitination assay as indicated. Samples were analyzed by immunoblotting with indicated antibodies. (B) Recombinant PARP1 was subjected into in vitro ubiquitination assay along with RNF168 WT or its point mutants as indicated. Black and red star indicate PARP1 and RNF168, respectively. (C) Validation of siRNAs and siRNA resistant (siRe) mutants of RNF168. (D) The intra-cellular level of PARP1 was monitored by transfection with RNF168 WT and its mutants as indicated. (E) The change of PARP1 level in the chromatin fraction was analyzed by overexpression with HA-ubiquitin and RNF168 as indicated. Black star indicates endogenous PARP1. (F) Chromatin fraction was subjected into in vitro ubiquitination assay as indicated. RNF168 mediated PARP1’s ubiquitination was monitored by immunoblotting with anti-GST and PARP1 antibodies. Black star indicates intact PARP1 in the chromatin fraction. F.S. represents fragment spectrum of GST-RNF168 (G) RNF168 induces PAR dependent degradation of PARP1 by ubiquitination in a chromatin context-dependent manner. PARP1’s protein level regulated by RNF168 was monitored in absence or presence of MG132 as indicated. (H) RNF168 mediated PARP1’s ubiquitination sites were analyzed by a mass-spectrometry analysis. Bold type K letters as marked red color indicate ubiquitinated lysine residues on PARP1 or ubiquitin. Ub(n), denotes polyubiquited chains of target proteins; RNF168 Ub(n) or PARP1 Ub(n) represents ubiquitinated RNF168 or PARP1, respectively.
Mol. Cells 2018;41:799~807 https://doi.org/10.14348/molcells.2018.0078
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