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. 2012 Apr 16;197(2):179–187. doi: 10.1083/jcb.201106152

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© 2012 Danielsen et al.

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Figure 1. — PIAS4- and RNF8-dependent SUMOylation of HERC2 and RNF168 in response to DSBs. (A) HeLa/FLAG-SUMO cell lines left untreated or induced to express SUMO1 or SUMO2 by addition of doxycycline (DOX) for 24 h were subjected to IR or not and harvested 1 h later. Cells were lysed under denaturing conditions, and protein SUMOylation was analyzed by immunoblotting of FLAG IPs with the indicated antibodies. MM, molecular mass; WCE, whole-cell extract. (B) HeLa/FLAG-SUMO1 cells exposed to IR or UV were processed as in A. (C) HeLa/FLAG-SUMO1 cells transfected with control (CTRL), PIAS1, or PIAS4 siRNAs 24 h before addition of doxycycline were processed as in A. Knockdown efficiency of PIAS1 and PIAS4 siRNAs is shown in Fig. S1 E. (D) U2OS cells transfected or not with HA-tagged PIAS4 plasmid for 24 h were exposed to IR and harvested 1 h later. Cell extracts were subjected to HA IP followed by immunoblotting. (E) HeLa/FLAG-SUMO1 cells transfected with control or RNF8 siRNAs for 24 h were processed as in A.