PARP1 Trapping Assay Protocol

PARP1 trapping assay was adapted from (Murai et al, 2012 (link)). In brief, 24 h prior the assay, KB2P and KB2P PARG ko cells were seeded on 10-cm dishes to achieve ~90% confluency. Cells were treated with 500 nM olaparib or 10 µM LNT1 or 1 µM PDDX-004 for 2 h, with the last 30 min in the presence of 0.01% MMS, as indicated. After the treatments cells were trypsinized and subsequently lysed to isolate chromatin-bound fractions. Fractionation was performed with Subcellular Protein Fractionation Kit from Thermo Scientific (#78840, Rockford, IL, USA), following the manufacturer’s instructions. Immunoblotting was carried out as described in the previous section (PAR Immunoblotting), using the rabbit polyclonal anti-PARP1 (#9542, Cell Signaling) primary antibody in dilution 1:1000 and the secondary goat polyclonal anti-rabbit immunoglobulins/HRP (Dako), diluted 1:5000.