Fig. 3: USP10 deubiquitinates PARP1 at the K418 site. | Oncogene

Fig. 3: USP10 deubiquitinates PARP1 at the K418 site.

From: The deubiquitination-PARylation positive feedback loop of the USP10-PARP1 axis promotes DNA damage repair and affects therapeutic efficacy of PARP1 inhibitor

Fig. 3

A PARP1 ubiquitination level determined by immunoblotting analysis. Lysates prepared from HEK293 cells transfected with or without Flag-USP10 for 48 h were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. B In vitro de-ubiquitination assay to determine the direct effect of USP10 on PARP1 polyubiquitination. Myc-PARP1, Flag-USP10 and HA-Smurf2 were purified from HEK293 cells. These purified proteins were incubated with Mg-ATP, Ubiquitin, E1 and E2 at 37 °C for 1 h. After termination, the reaction was subjected to immunoblotting with anti-ubiquitin antibody. C PARP1 ubiquitination in HEK293 cells transfected with or without USP10 siRNA for 48 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. D PARP1 ubiquitination in HCT116 cells with or without USP10 knockdown. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. E PARP1 ubiquitination in MCF7 cells treated with or without Spautin-1 (30 μM) for 24 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. F The potential ubiquitination sites of PARP1 protein identified by mass spectrometry. G Western blot analysis of lysates prepared from HEK293 cells co-transfected with wild-type or mutant Flag-PARP1 (K418R) and control (Myc) or Myc-USP10 expression plasmids, as indicated, for 48 h. Data are presented as the mean ± SEM from three independent experiments using the Student’s t test. **P < 0.01, N.S. not significant. H–M Co-IP analysis of PARP1 wild-type and mutant proteins. HEK293 cells were co-transfected with HA-ubiquitin, Myc-USP10, and Flag-PARP1 wild-type, Flag-PARP1 K418R, Flag-PARP1 K131R, Flag-PARP1 K249R, Flag-PARP1 K548R, Flag-PARP1 K579R or Flag-PARP1 K633R plasmids for 48 h. Lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-HA antibody.

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