Extended Data Fig. 6: Trapped PARP1 interacts with p97.
From: The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin
a. Western blot analysis of Co-IP confirms PARP1–p97 interaction. CAL51 cells were transiently transfected with p97-WT-GFP-expressing construct. Subsequently, GFP was immunoprecipitated in native conditions and the presence of PARP1 investigated by Western blotting. Data shown represent 2 biological replicas. b. PARP1 interacts with p97 in a trapping-dependant manner. Cells were treated with 0.01% MMS in the presence of 100 nM talazoparib, 10 µM veliparib or 10 µM UKTT15. PARP1 associated proteins were immunoprecipitated and the presence of p97 was investigated by immunoblotting. Data shown represent 2 biological replicas. c. Western blots for denaturing IP experiment shown in Fig. 4f. Data shown represent 3 biological replicas. d. p97 E578Q mutant colocalises with PARP1 under trapping conditions. CAL51 PARP1WT-eGFP and PARP1del.p.119K120S-eGFP cells were transfected with p97-WT-Strep-MYC or p97 E578Q-Strep-MYC constructs and then subsequently exposed to MMS + talazoparib to induce PARP1 trapping. Cell were then pre-extracted and fixed, and stained for trapped PARP1 and MYC (as described in34). The p97 E578Q-mutant colocalised with the trapped PARP1 signal in CAL51 PARP1WT-eGFP cells (yellow arrows) whereas PARP1del.p.119K120S-eGFP were unable to form trapped PARP1 foci. Scale bar = 5 µm. Data shown represent 2 biological replicas. e. Ubiquitin is required for the PARP1/p97 interaction in trapping conditions. Western blots of PARP1 co-immunoprecipitates from CAL51 PARP1WT-eGFP-expressing cells. Trapping increases the PARP1/p97 interaction (lane 4), an effect reversed by MLN-7243 (5 μM). Data shown represent 3 biological replicas.
