t4 dna ligase  (Becton Dickinson)

Journal: Neurobiology of aging

Article Title: Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice

doi: 10.1016/j.neurobiolaging.2015.04.004

Figure Lengend Snippet: Expression of Xrcc1 DNA Ligase III and whole BER activity in Xrcc1 +/− mouse brains after middle cerebral artery occlusion/ reperfusion (MCAO/R). (A) A representative immunoblot of Xrcc1 and Ligase III levels in whole brain lysates of

Article Snippet: Primary antibodies used were: anti-Xrcc1 (Cell Signaling, Cat # 2735S); anti-NEIL1 (Santa Cruz, SC-134547); anti-DNA ligase III (BD Biosciences, Cat # 611876); anti-Caspase-3 (Cell Signaling, Cat #9662); and anti-β-actin (Santa Cruz, SC-1616).

Techniques: Expressing, Activity Assay

Journal: Nature

Article Title: Regulation of DNA Repair pathway choice in S/G2 by the NHEJ inhibitor CYREN

doi: 10.1038/nature24023

Figure Lengend Snippet: CYREN prevents cNHEJ in S and G2 at deprotected telomeres. a, Schematic of CO-FISH. Chromatid-type fusions involving leading and lagging strands. b, Percentage of fusions ± upper and lower value of 95% confidence intervals, Wilson/Brown test. 126 fusions counted. c, Western blot showing knockdown of ATM, Ligase 4, DNA-PKcs and Ligase 3 in Figure 2c . For gel source data, see Supplementary Figure 1 . d, Experimental timeline for Figure 2c . CYREN wt and CYREN KO HT1080 cells were infected with shTRF2 on Day 0, followed by transfection with siRNAs on Day 2. On Day 3, shTRF2 infected cells were selected with Puromycin and cells were collected for fusion analysis on Day 5. e, Experimental timeline for Extended data Figure 3d . HT1080 6TG were stably transduced with shTRF2 on Day 0, followed by transfection with Non-Targetting (NT) or CYREN siRNAs on Day 2. On Day 3, shTRF2 infected cells were selected with Puromycin and inhibitors were added. Cells were collected for fusion analysis on Day 5. f, Percentage of cells with fusions ± upper and lower value of 95% confidence intervals, Wilson/Brown test. Cells were treated for 48 hours with DMSO or the following inhibitors: ATMi (KU-55933) 10µM, DNA-PKcsi (NU-7441) 1µM, PARPi (Olaparib) 10µM, RAD51i (RI-1) 20µM. ****P

Article Snippet: Antibodies: TRF2 (Karlseder lab), Rabbit FLAG (Sigma-Aldrich - F7425), FLAG (M2, Sigma-Aldrich - F1804), Tubulin (Sigma-Aldrich – T6557), BrdU A488 (3D4, BD Biosciences - 555627), Ku70 (V540, Cell Signalling - 4104), Ku70 (Abcam – ab3114), Ku86 (Cell Signalling - 2753), ATM (Epitomics - 1549-1), Ligase 4 (EPR16531, Abcam - ab193353), DNA-PKcs (Abcam - ab70250), Ligase 3 (BD Biosciences - 611876), Anti-Rabbit HRP (GE Healthcare - NXA931), Anti-Mouse HRP (GE Healthcare - NXA934V).

Techniques: Fluorescence In Situ Hybridization, Western Blot, Infection, Transfection, Stable Transfection, Transduction

Journal: Nucleic Acids Research

Article Title: DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

doi: 10.1093/nar/gku1307

Figure Lengend Snippet: PARP1 inhibition and the recruitment of SSBR proteins to sites of DNA damage. HeLa cells expressing fluorescently tagged SSBR proteins were treated with the PARP inhibitor, AG14361, and then subjected to laser micro-irradiation. PARP inhibition only affected early recruitment events of ( A ) XRCC1, ( B ) PNKP and ( C ) LIG3 with almost no effect on the late events of accumulation of all the proteins at sites of DNA damage. For recruitment curves, error bars represent SEM from three independent experiments for a total of 36 individual cells.

Article Snippet: The antibodies used included mouse monoclonal anti-DNA ligase 3 (cat. no. 611876, BD Transduction Labs, Mississauga, ON, USA), goat polyclonal anti-actin (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Inhibition, Expressing, Irradiation

Journal: Nucleic Acids Research

Article Title: DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

doi: 10.1093/nar/gku1307

Figure Lengend Snippet: Recruitment and retention of SSBR proteins following multi-photon 750-nm laser micro-irradiation. EGFP-PARP1, XRCC1-mGFP and EGFP-LIG3 show near instantaneous recruitment to sites of DNA damage, and PNKP-mGFP is also rapidly recruited. Laser micro-irradiation using multi-photon 750 nm was carried out as outlined in the Materials and Methods section using HeLa cells expressing fluorescently tagged versions of indicated proteins. Recruitment curves show quantification of signals over the observed time scale starting at the time when the damage is introduced by the laser ( t = 0). Error bars represent SEM from three independent experiments for a total of 36 individual cells.

Article Snippet: The antibodies used included mouse monoclonal anti-DNA ligase 3 (cat. no. 611876, BD Transduction Labs, Mississauga, ON, USA), goat polyclonal anti-actin (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Irradiation, Expressing

Journal: Nucleic Acids Research

Article Title: DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

doi: 10.1093/nar/gku1307

Figure Lengend Snippet: LIG3 knockdown and the recruitment of XRCC1 and PNKP to sites of DNA damage. Laser micro-irradiation was performed on HeLa cells expressing reduced levels of LIG3 (see Supplementary Figure S5) and ( A ) XRCC1-mRFP or ( B ) PNKP-mRFP. Reduced background levels of LIG3 lead to decreased overall recruitment of XRCC1 and PNKP to sites of DNA damage. ( C ) Simultaneous inhibition of PARP1 (using AG14361) and knockdown of LIG3 showed an additive effect on the reduction of the amount of PNKP recruited to sites of DNA damage. For recruitment curves, error bars represent SEM from three independent experiments for a total of 36 individual cells. Note that the mRFP photobleaches during laser micro-irradiation resulting in an initial loss of fluorescence at the damage sites.

Article Snippet: The antibodies used included mouse monoclonal anti-DNA ligase 3 (cat. no. 611876, BD Transduction Labs, Mississauga, ON, USA), goat polyclonal anti-actin (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Irradiation, Expressing, Inhibition, Fluorescence