par inhibitor  (MedChemExpress)

Journal: FEBS open bio

Article Title: Interaction of RECQL4 with poly(ADP-ribose) is critical for the DNA double-strand break response in human cells.

doi: 10.1002/2211-5463.13917

Figure Lengend Snippet: Fig. 1. The recruitment of RECQL4 to DSB sites requires the activity of PARP-1. (A, B) U2OS or PARP1-KO-U2OS cells were transfected with EGFP-RECQL4. EGFP-RECQL4 transfected U2OS cells were mock-treated (DMSO) or treated with an inhibitor of PARP (PARPi, 10 lM olaparib), ATM (ATMi, 10 lM KU-55933), DNA-PK (DNA-PKi, 10 lM NU7441), or PARG (PARGi, 1 lM PDD00017273) for 1 h, and EGFP- RECQL4 binding to microirradiation sites was determined. Representative images (A) and graphs (B) are shown. (C, D) EGFP-tagged RECQL4 derivatives were expressed in U2OS cells and their binding to laser microirradiation sites was determined. Representative images (C) and graphs (D) are shown. HT, helicase truncated RECQL4; WA, Walker A mutant; WB, Walker B mutant; WT, wild-type. The scale bar represents 10 lm. Data in graphs are means SEM; n = 15 from three independent experiments.

Article Snippet: The PAR polymerase (PARP) inhibitor, olaparib, was purchased from Selleckchem.

Techniques: Activity Assay, Transfection, Binding Assay, Mutagenesis

Journal: FEBS open bio

Article Title: Interaction of RECQL4 with poly(ADP-ribose) is critical for the DNA double-strand break response in human cells.

doi: 10.1002/2211-5463.13917

Figure Lengend Snippet: Fig. 2. The small region of RECQL4 sufficient for rapid recruitment to DSB sites directly interacts with PAR. (A) Schematic diagram of truncated RECQL4 proteins and their binding to laser-induced DSB sites and graphs. NLS: the nuclear localization signal sequence of Simian virus 40. Data in graphs are means SEM; n = 15, from three independent experiments. (B) Recruitment of the region of RECQL4 (aa 360–437) to DSB sites was determined in cells treated with a PARP inhibitor (olaparib) or a PARG inhibitor (PDD000172273). The scale bar represents 10 lm. Data in graphs are means SEM; n = 15, from three independent experiments. (C) Co-precipitation of RECQL4 proteins by PAR immunoprecipitation (IP) in cells treated as indicated. Anti-PAR antibodies were used in IP, and 10% of the extract for IP was used in input lanes. Bleo, bleomycin; (), untreated. (D) GST and GAT-RECQL4360–437 (GST-R4360–437) proteins were expressed in Escherichia coli cells and purified. (E) The PAR polymer (20 pmol) was incubated with the indicated amount of GST or GST-R4360–437 protein, and GST pull- down assays were carried out. For PAR control, the indicated amount of PAR polymer was directly immobilized onto a nylon membrane. Immunoblotting was carried out with an anti-PAR antibody. (F) For the PAR pull-down assay, 30 pmol of biotin-labeled PAR polymer was incubated with 30 pmol of GST or GST-R4360–437 protein and streptavidin beads. Western blotting was carried out with an anti-GST antibody. In the input lanes, 25% of input materials were used. (C, E, F) These experiments were conducted more than once and the results of a single experiment are shown.

Article Snippet: The PAR polymerase (PARP) inhibitor, olaparib, was purchased from Selleckchem.

Techniques: Binding Assay, Sequencing, Virus, Immunoprecipitation, Polymer, Incubation, Control, Membrane, Western Blot, Pull Down Assay, Labeling

Journal: FEBS open bio

Article Title: Interaction of RECQL4 with poly(ADP-ribose) is critical for the DNA double-strand break response in human cells.

doi: 10.1002/2211-5463.13917

Figure Lengend Snippet: Fig. 3. The putative PAR-binding motif (PBM) in RECQL4 is important for the recruitment of RECQL4 to DSB sites. (A) Schematic diagram of human RECQL4, the amino acid sequence of the putative PBM in RECQL4, and its replacement with wild-type (RECQL4-hnPAR) or mutated PBM (RECQL4-hnPARm) in hnRNPA2. Substituted amino acid positions are indicated as a gray background, and the positions replaced with alanine are indicated in red. Numbers refer to the amino acid positions from the initiation codon. (B) Recruitment of various RECQL4 derivatives to laser-induced DSB sites in the absence or presence of a PARP inhibitor (olaparib). Representative images (left panel) and graphs (right panel) are shown. R4 stands for RECQL4. The scale bar represents 10 lm. Data in graphs are means SEM; n ≥20, from three independent experiments.

Article Snippet: The PAR polymerase (PARP) inhibitor, olaparib, was purchased from Selleckchem.

Techniques: Binding Assay, Sequencing