Fig. 4: PARP1 mediates PARylation of USP10 at D634, D645, E648 site. | Oncogene

Fig. 4: PARP1 mediates PARylation of USP10 at D634, D645, E648 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. 4

A PARylation of USP10 determined by immunoprecipitation analysis in HEK293 cells. Lysates were immunoprecipitated with control IgG or anti-USP10 antibody, followed by immunoblotting with the indicated antibodies. B HEK293 cell lysates were immunoprecipitated with control IgG or anti-PAR antibody and then immunoblotted with the indicated antibodies. C In vitro Poly (ADP-ribose) (PAR) binding assay of USP10. Bacterially produced re-combinant GST-USP10 was used to pull down Biotin PAR-polymer (20 pmol) followed by dot blot analyses. USP10 PARylation level was determined in HEK293 cells that were transfected with Myc or Myc-PARP1 plasmids for 48 h (D), treated with or without PARP1-siRNA for 48 h (E), treated with or without 10 mM Olaparib for 24 h (F), or treated with or without 10 mM PARG inhibitor for 24 h (G). Lysates were immunoprecipitated with anti-USP10 antibody, followed by immunoblotting with anti-PAR antibody. H The PARylation level of the USP10 truncations was determined in HEK293 cells transfected with the indicated Flag-USP10 expression plasmids. I HEK293 cells were transfected with Flag-USP10 wild-type, Flag-USP10 (K620A, E621A, D634A, D645A, E648A, E654A, K687A, E692A, K693A and K699A) plasmids for 48 h. Lysates were immunoprecipitated with anti-Flag antibody, and the level of PARylation was determined by immunoblotting with an anti-PAR antibody.

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