Skip to main content
. 2014 Jan 31;42(7):4435–4449. doi: 10.1093/nar/gku088

Figure 5.

Figure 5.

PARP1 recruits both XRCC1 and TDP1 to Top1-induced damage sites. (A) Endogenous PARP1 co-immunoprecipitates TDP1. HCT116 cells were treated with CPT (10 µM for 2 h) alone or with ABT-888 (5 µM for 2 h). Endogenous PARP1 was immunoprecipitated with anti-PARP1 antibody and immune complexes were blotted with anti-PAR or anti-TDP1 antibodies. Blots were subsequently stripped and probed with anti-PARP1 antibody. Control immunoprecipitation with anti-IgG antibody demonstrates the specificity of the reactions. (B) TDP1 co-immunoprecipitates PARP1. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells. Following CPT treatment without or with ABT-888 (as in A), TDP1 was immunoprecipitated with anti-Flag antibody and immune complexes were probed with anti-PAR, anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Protein molecular weight markers (kDa) are indicated at right. (C) Knocking down PARP1 abrogates the TDP1-XRCC1 interaction. Flag-tagged hTDP1 was ectopically expressed in isogenic HeLa cells stably transfected with PARP1-shRNA or control (Ctr)-shRNA. Following CPT treatment (as in A), ectopic TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were blotted with anti-XRCC1 antibodies. (D) TDP1–PARP1 association is not mediated through DNA. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells treated with CPT (as in A). Cell lysates was pretreated with Benzonase® nuclease before co-immunoprecipitation. The immune complexes were probed with anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Migration of protein molecular weight markers (kDa) is indicated at right. Aliquots (10%) of the input show the level of indicated proteins before immunoprecipitation. (E) CPT-induced XRCC1 foci are abrogated by ABT-888. Representative immunofluorescence microscopy image of HCT116 cells treated with CPT (1 µM for 3 h) alone or with ABT-888 (5 µM for 3 h). Focal accumulation of PAR-polymers and XRCC1 are shown in green. (F) Defective XRCC1 focus formation in HeLa cells stably transfected with PARP1-shRNA. Representative immunofluorescence microscopy image of control-shRNA (Ctr) or PARP1-shRNA HeLa cells were treated with CPT (1 µM for 3 h) and subsequently fixed and immunostained for XRCC1 (green) or PARP1 (red). Nuclei are outlined as dashed white line circle.