Ubiquityl pcna lys164

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).