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. Author manuscript; available in PMC: 2015 Nov 4.
Published in final edited form as: Cell Metab. 2014 Nov 4;20(5):840–855. doi: 10.1016/j.cmet.2014.10.005

Fig. 6. CSB inhibits PARP1 activation in vitro and in vivo through displacement of PARylated PARP1 from DNA.

Fig. 6

(A) Electromobility shift assay (EMSA) of PARP1 and CSB binding to double stranded DNA. (B) BamH1 incision of a 42-mer oligo preincubated with 250 nM PARP1 and with increasing amounts of CSB with or without NAD+ (n=3, mean ± SEM). (C) Representative immunoblot and quantification of in vitro poly-ADP-ribosylation (PARylation) of CSB and PARP1. Reactions were performed with recombinant proteins in the presence of undamaged or damaged DNA (n=3, mean ± SEM). (D) Representative immunoblot of whole cell (PARylation) after 5 J/m2 treatment in WT and CSB deficient cells at various timepoints. (E) Recruitment of gfp-tagged CSB to laser induced DNA damage after 1 hour preincubation with PARP inhibitors 3AB or PJ34. (F) Representative slot-blot showing non-covalent interaction between CSB and PAR. (G) The PAR binding motif in CSB. (H) Recruitment to laser induced DNA damage of gfp-tagged WT CSB or CSB harboring K to A mutations in the 4 conserved lysines. (I) Recruitment of gfp-tagged WT CSB to laser induced DNA damage in WT HeLa or PARP1−/− HeLa cells.