TARG1-type macrodomain PAR hydrolysis was determined by western blot33 (link). The substrate used was automodified hPARP obtained as described above using NAD+ and activated DNA as substrates and Rucaparib to stop the reaction. Then, this PARylated substrate was incubated for 1 hour at 30 °C in the presence of FmTARG1 (20 μM) or hTARG1 (10 μM), before running the samples on 7–10% SDS-PAGE gels and transfering to a nitrocellulose membrane. The presence of PAR was detected by coupling with rabbit polyclonal anti-PAR antibodies (1:1000, Trevigen) and goat-anti-rabbit-HRP conjugated secondary antibody (Bio-Rad), and finally revealed with Opti-4CN substrate (Bio-Rad) for 5 minutes. Analysis of the reaction products of hTARG1 and FmTARG1 on poly (ADP-ribosyl)ated PARP1 was carried out by mass spectrometry as described above for the de-MARylation assay.
Anti par antibody
Manufactured by Bio-Techne
Sourced in United States
The Anti-PAR antibody is a laboratory reagent used for the detection and quantification of poly(ADP-ribose) (PAR) in biological samples. PAR is a post-translational modification involved in various cellular processes, and the Anti-PAR antibody can be used to study its roles and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Subcellular protein fractionation kit, by Thermo Fisher Scientific (2 mentions)
Opti 4cn substrate, by Bio-Rad (1 mentions)
Goat anti rabbit hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Opti 4cn, by Bio-Rad (1 mentions)
Anti biotin, by Merck Group (1 mentions)
Anti rpa32, by Cell Signaling Technology (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Cisplatin, by J&K Scientific (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Olaparib, by Targetmol (1 mentions)
Anti rad51, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Anti parp antibody, by Cell Signaling Technology (1 mentions)
Rad51, by Cell Signaling Technology (1 mentions)
Ercc1, by Santa Cruz Biotechnology (1 mentions)
Acetyl h3, by Cell Signaling Technology (1 mentions)
Anti uhrf1, by Santa Cruz Biotechnology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
8 protocols using anti par antibody
1
TARG1-mediated PAR hydrolysis assay
2
Quantifying PARP Automodification and PARG-Mediated PAR Hydrolysis
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PARP automodification was also assayed by western blot in a reaction containing different concentrations of enzyme (250 nM HaPARP, 200 nM CdPARP, or 85 nM hPARPl) in assay buffer, with or without NAD+ (1 mM), activated DNA or rucaparib (1 µM), respectively. The reactions were allowed to proceed for 1 h at 25 °C. PAR-mediated PARG hydrolysis was analysed using the above-mentioned PARP automodification reaction after stopping it with rucaparib, followed by the addition of PARG enzymes (500 nM) and incubation for 1 hour at 30 °C. The above reactions were then run on 7–10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The presence of poly ADP-ribose (PAR) was detected by the use of rabbit polyclonal anti-PAR antibodies (1:1000; Trevigen), and goat anti-Rabbit IgG antibody conjugated to horseradish peroxidase (1:5000; Bio-Rad), and Opti-4CN as optimized colorimetric substrate (Bio-Rad).
3
Characterizing EXO1 Interactions and Variants
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GFP-EXOI plasmids were a gift from Zhongsheng You. N-terminus (a.a. 1–352), middle region (a.a. 353–549) and C-terminus (a.a .550–846) of EXO1, SMG5-PIN (a.a. 831–1016), GEN1-PIN (a.a. 1–210) were cloned into pEGFP-C1 or pGEX-4T vector. The EXO1 natural variants and siRNA resistant forms were generated using the QuikChange site-directed mutagenesis kit (Stratagene).
The siRNA sequences targeting PARP1, PARP2, MSH3 and EXO1 are 5′-CAAAGUAUCCCAAGAAGUUdTdT-3′, 5′-GGAGAAGGAUGGUGAGAAAdTdT-3′, 5′-GAAGAACAAUAUCCUACUAdTdT-3′ and 5′-CAAGCCUAUUCUCGUAUUUdTdT-3′ respectively. siRNAs were transfected into cells using oligofectamine (Invitrogen) according to manufacturer's instructions.
Anti β-actin, anti-biotin, anti-EXO1 and anti-GFP antibodies were purchased from Sigma. Anti-PARP1 and anti-PARP2 antibodies were purchased from Millipore. Anti-PAR antibody was purchased from Trevigen. Anti- RPA32 and phospho-H2AX (γ-H2AX) were purchased from Cell Signaling.
The siRNA sequences targeting PARP1, PARP2, MSH3 and EXO1 are 5′-CAAAGUAUCCCAAGAAGUUdTdT-3′, 5′-GGAGAAGGAUGGUGAGAAAdTdT-3′, 5′-GAAGAACAAUAUCCUACUAdTdT-3′ and 5′-CAAGCCUAUUCUCGUAUUUdTdT-3′ respectively. siRNAs were transfected into cells using oligofectamine (Invitrogen) according to manufacturer's instructions.
Anti β-actin, anti-biotin, anti-EXO1 and anti-GFP antibodies were purchased from Sigma. Anti-PARP1 and anti-PARP2 antibodies were purchased from Millipore. Anti-PAR antibody was purchased from Trevigen. Anti- RPA32 and phospho-H2AX (γ-H2AX) were purchased from Cell Signaling.
4
Targeted Cancer Therapy Compound Evaluation
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YHP-836 was synthesized in-house. PARP1/2 inhibitor olaparib was purchased from TargetMol, United States. Temozolomide (TMZ), topotecan, cisplatin, and adriamycin were purchased from J&K Scientific (Beijing, China). Anti-γH2AX and anti-RAD51 were obtained from Cell Signaling Technology (Danvers, MA, United States). An anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Anti-PARP1 and anti-PARP2 antibodies were from Abcam (Cambridge, United Kingdom). Anti-PAR antibody and HT PARP pharmacodynamic assay kit were purchased from Trevigen (Gaithersburg, MD, United States). The subcellular protein fractionation kit was purchased from Thermo Scientific (Rockford, IL, United States).
5
Immunoprecipitation and Western Blotting
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Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride). Cell lysate (200-1000 µg of protein) was immunoprecipitated with anti-PARP antibody (Cell Signaling, Boston, MA), anti-MGMT antibody (Sigma), or anti-PAR antibody (Trevigen) for 18 hours at 4°C. Normal preimmune mouse and rabbit IgG (Cell Signaling Technology, Boston, MA) was also used as negative controls. The immunoprecipitates were eluted by the 2× SDS sample buffer and then detected by Western blot analysis.
6
Histone PARylation Detection in U2OS Cells
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U2OS cells were lysed with NETN-100 buffer (50 mM Tris–HCl pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) on ice for 30 min. Soluble fractions were subjected to immunoprecipitation and dot blotting and probed with anti-PAR antibody (Trevigen). For detecting histone PARylation, cells were lysed with 0.5% SDS and the lysates were diluted to 0.1% SDS for immunoprecipitation with anti-histone antibodies. The samples were subjected to dot blotting assay using anti-PAR antibody. To validate the recognition of SSRP1 to PARylated histone, cells were lysed with NETN-100 buffer and the lysates were immunoprecipitated with anti-SSRP1 or anti-FLAG antibodies. The immunoprecipitated proteins were eluted from the beads by 0.5% SDS and diluted to 0.1% SDS for the second round of immunoprecipitation with the anti-PAR antibody and blotting with anti-H2B antibody.
7
Protein Expression Analysis by Western Blot
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Protein levels were assessed by western blot. Antibodies against HDAC, PARP, cleaved-PARP, H3, Acetyl-H3, and RAD51 were purchased from Cell Signaling Technology (CST). Anti-UHRF1, Ku-70, ERCC1, MSH2, MSH6, GAPDH and anti-α-tublin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRCA1 and phosphorylated-BRCA1(ser988) was purchased from ABclonal Technology (Upper Heyford, UK). Anti-PAR antibody was purchased from Trevigen (4335-MC-100, MD, USA).
8
PARP-2 Automodification and DNA Binding Assay
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PARP-2 WT (60 nM) was incubated with 60 nM DNA (dnick or dnick 5′P) for 30 min at
RT in the presence of 25 μM NAD+ in the same buffer conditions as the
radioactive and colorimetric assays. In Supplementary Figure S6, PARP-2 WT and L269A (60
nM) were incubated with 25 μM NAD+ in the absence of DNA. Reactions were
stopped by the addition of SDS-PAGE loading buffer, incubated for 5 min at 95°C,
resolved on 10% SDS-PAGE (50 ng of protein) and blotted onto Hybond-ECL
Nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h (RT) in the following
buffer: tris-buffered saline with tween (TBST; 20 mM Tris pH 7.5, 150 mM NaCl, 0.1%
Tween 20) supplemented with 5% blocking-grade blocker (Bio-Rad). Blots were
incubated 1 h (RT) with a 1:2000 anti-PAR antibody (Trevigen) in blocking buffer and
washed with TBST and TBS then incubated with 1:7000 HRP conjugated donkey anti-rabbit
antibody (Santa Cruz Biotechnology) in 1% blocking-grade buffer in TBST. Blots were
washed and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific).
RT in the presence of 25 μM NAD+ in the same buffer conditions as the
radioactive and colorimetric assays. In Supplementary Figure S6, PARP-2 WT and L269A (60
nM) were incubated with 25 μM NAD+ in the absence of DNA. Reactions were
stopped by the addition of SDS-PAGE loading buffer, incubated for 5 min at 95°C,
resolved on 10% SDS-PAGE (50 ng of protein) and blotted onto Hybond-ECL
Nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h (RT) in the following
buffer: tris-buffered saline with tween (TBST; 20 mM Tris pH 7.5, 150 mM NaCl, 0.1%
Tween 20) supplemented with 5% blocking-grade blocker (Bio-Rad). Blots were
incubated 1 h (RT) with a 1:2000 anti-PAR antibody (Trevigen) in blocking buffer and
washed with TBST and TBS then incubated with 1:7000 HRP conjugated donkey anti-rabbit
antibody (Santa Cruz Biotechnology) in 1% blocking-grade buffer in TBST. Blots were
washed and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific).
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