Extended Data Fig. 2: Purification of PARP1 From HEK293T cells and characterization of biochemical activity.
From: BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments
(a) Coomassie blue stain and western blot of purified full-length PARP1. Lane 1,4: Marker, lane 2: 2XMBP-PARP1, lane 3: purified PARP1, lane 5: western blot for PARP1. (b) In vitro PARylation assay using recombinant PARP1 in the presence of NAD+ and an activator oligonucleotide (0.2 µM) mimicking DNA strand break. Stain free gel shows PARP1 loading in all lanes. (c) In vitro PARylation inhibition assay using recombinant PARP1 in the presence of NAD+, activator oligonucleotide and PARPi. (d) PARylated PARP1 dissociation assay using PARP1 and NAD+ and 40 bp ds-DNA (0.4 nM). (e) EMSA analyses of PARP1 binding to 0.4 nM of dsDNA and 3′ tailed DNA. (f) EMSA analyses of PARP1 binding to 0.4 nM of ss-DNA. (g) Quantification of EMSA results from e and f. Data shown is mean ± s.d (r = 3).
