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. 2021 Sep 11;49(18):10477–10492. doi: 10.1093/nar/gkab771

Figure 5.

Figure 5.

Detection of thymidine-linked ADP-ribosylation in human genomic DNA. (A) U-2 OS TARG1 KO cells expressing GFP-DarT (24 h) or treated with MMS (2 mM, 1 h) were also treated with olaparib (10 μM, 24 h) or veliparib (10 μM 24 h). Levels of KAP1 pS824, RPA2 pS4/8, γH2AX and ADP-ribosylation were assessed. (B) Representative images of U-2 OS TARG1 KO cells treated as in (A). Cells were pre-extracted and immunostained for GFP, ADP-ribosylation and chromatin-bound RPA2. Scale bar 10 μm. (C) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Genomic DNA was extracted and dotted onto nitrocellulose membranes and immunoblotted for dsDNA or ADP-ribosylation using the CST poly/mono ADP-ribose antibody. (D) Genomic DNA from U-2 OS TARG1 KO cells expressing DarT was extracted as in (C). DNA was incubated with either 1 μM TaqDarG-macrodomain WT or TARG1 WT recombinant proteins in vitro. DNA was then immunoblotted as in (C).