Pol η

The proteins in the NEs/whole cell extracts were separated onto a Bio-Rad 4 to 20% gradient Bis-Tris gel, then electrotransferred on a nitrocellulose (0.45 μm pore size; GE Healthcare) membrane using 1× Bio-Rad transfer buffer. The membranes were blocked with 5% w/v skimmed milk in TBST buffer (1× Tris-Buffered Saline, 0.1% Tween 20), then immunoblotted with appropriate antibodies (Pol η [Cell Signaling Technology Cat# 13848S], PNKP, γ-H2AX [ser phos 139 residue; Cell Signaling Technology Cat# 9718S], unmodified H2AX [Cell Signaling Technology Cat# 2595S], p53BP1 [S1778, Cell Signaling Technology Cat# 2675S], unmodified 53BP1 [Santa Cruz Biotechnology Cat# sc 517281], pATM [Santa Cruz Biotechnology Cat# sc 47739], unmodified ATM [Santa Cruz Biotechnology Cat# sc 377293], HDAC2 [GeneTex Cat# 109642]). The membranes were extensively washed with 1% TBST followed by incubation with anti-isotype secondary antibody (Cell Signaling Technology) conjugated with horseradish peroxidase in 5% skimmed milk at room temperature. Subsequently, the membranes were further washed three times (10 min each) in 1% TBST, developed, and imaged using Kwikquant image analyzer and image analysis software (ver 5.2) (Kindle Biosciences).

Human HeLa and RPE-1 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. For PARP10 gene knockout, the commercially available PARP10 CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-406703). Single cells were FACS-sorted into 96-well plates using a BD FACSAria II instrument. Following clonal expansion, resulting mono-clonal cultures were screened by western blot for PARP10 protein levels. For exogenous PARP10 expression, pLV-puro-TRE lentiviral constructs encoding wild-type or the ΔPARP variant spanning amino acids 1–834 (lacking the PIP motif and PARP catalytic domain), with the Myc-epitope tag EQKLISEEDL at the N-terminus, were obtained from Cyagen. Infected cells, stably expressing the tetracycline transactivator element, were selected by puromycin. For induction of expression, cells were grown in the presence of 2 μg/ml doxycycline. Cell extracts, chromatin fractionation and western blot experiments were performed as previously described (14 (link),23 (link),24 (link)). Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).