Anti cleaved parp

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, Germany, Switzerland

Anti-cleaved PARP is a laboratory product that detects the cleaved form of Poly(ADP-ribose) Polymerase (PARP), a protein involved in cellular processes such as DNA repair and programmed cell death. This product is used for the identification and analysis of the cleaved PARP fragment as a marker of apoptosis (programmed cell death) in various experimental systems.

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Lab products found in correlation

Close spelling variants of this product

Anti-PARP and cleaved PARP Anti-cleaved and total PARP Cleaved anti-human PARP Anti-cleaved PARP (D214), Rabbit anti-cleaved PARP Asp214 Rabbit anti-cleaved PARP Anti-cleaved PARP (9541) Anti-cleaved PARP (Asp214) rabbit monoclonal Ab (clone D64E10; Anti-cleaved poly ADP-ribose polymerase (PARP; Anti-cleaved PARP antibody

250 protocols using anti cleaved parp

Comprehensive Protein Analysis Protocol

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Western blotting was performed as described in [24 (link)]. Cell fractionation was performed as in [61 (link)]. The following primary antibodies were used: anti-Dicer, anti-cleaved PARP (detecting cleaved form of PARP), anti-cleaved PARP (detecting both full-length and cleaved PARP), anti-caspase-3, anti-caspase-9, anti-cleaved caspase-3, anti-cleaved caspase-9, anti-γH2AX, anti-cPLA₂ (Cell Signaling Technology, Inc., Danvers, MA, USA); mouse monoclonal anti-TSN (FIT Biotech, Tampere, Finland); anti-LC3 (MBL International Corporation, USA); anti-β-actin, anti-α-tubulin (Sigma-Aldrich, Stockholm, Sweden); anti-G3PDH (Trevigen Inc., Gaithersburg, MD, USA); anti-cytochrome c (BD Biosciences, San Jose, CA); anti-p62 and rabbit polyclonal anti-S100A11 Abs (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Protein molecular weights are presented on the right side of the corresponding western blotting panels. Densitometry of the protein signals was performed by using ImageJ software (http://rsbweb.nih.gov/ij/). The densitometry data were expressed as a fold change of the signal intensity compared to the lowest value, different from background readings, in the range of samples under comparison. The results shown are representative of three independent experiments.

Zagryazhskaya A., Surova O., Akbar N.S., Allavena G., Gyuraszova K., Zborovskaya I.B., Tchevkina E.M, & Zhivotovsky B. (2015). Tudor staphylococcal nuclease drives chemoresistance of non-small cell lung carcinoma cells by regulating S100A11. Oncotarget, 6(14), 12156-12173.

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Western Blot Analysis of Colon Tissue

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Colon tissues were lysed by using the Cell Lytic Buffer supplemented with both proteases (PIC, Merck) and phosphatases (NaF 10 mM; Na₃VO₄ 1 mM) inhibitors. Equal amounts of protein lysates (20 mg) were subjected to SDS-Page separation and proteins electroblotted onto Nitrocellulose (Merck) membranes. Non-fat 5% dry milk (Merck) in PBS was used as a blocking solution, and indicated primary antibodies, in blocking solution were incubated o.n. at 4 °C. Appropriate HRP-conjugated secondary antibodies were diluted in blocking solution (1:5000) and incubated for 1 h at r.t. A Westar ANTARES ECL kit (Cyanagen, Bologna, Italy) was then used and the signal acquired by a ChemiDoc^TM Touch (Bio-Rad), and analyzed using Image Lab software (5.0; Bio-Rad). Primary antibodies were as follows: anti-Bip/Grp78 (1:500; Santa Cruz, Dallas, TX, USA); anti-Calnexin (1:500; Santa Cruz); anti-Calreticulin (1:1000; Abcam, Cambridge, UK); anti-PARP cleaved (1:500; Cell Signaling, Danvers, MA, USA); anti-SLC7A11 (1:250; Cell Signaling); anti-Occludin (1:500; Cell Signaling); anti-Caspase-3 (1:250; Santa Cruz); anti-aActin (1:5000; Sigma, Darmstadt, Germany); anti-bTubulin (1:5000; Santa Cruz). HRP-conjugated secondary antibodies were diluted in blocking solution (1:5000; Jackson ImmunoResearch, Cambridgeshire, UK).

Monzani R., Gagliardi M., Clemente N., Saverio V., Pańczyszyn E., Santoro C., Yissachar N., Visciglia A., Pane M., Amoruso A, & Corazzari M. (2022). The Gut-Ex-Vivo System (GEVS) Is a Dynamic and Versatile Tool for the Study of DNBS-Induced IBD in BALB/C and C57BL/6 Mice, Highlighting the Protective Role of Probiotics. Biology, 11(11), 1574.

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Antibody Immunoprecipitation and Immunoblotting Protocol

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The following antibodies were used in for protein immunoprecipitation or immunoblotting: mouse anti-Flag M2 (Sigma), rabbit anti-Flag (Sigma), anti-GAPDH (Calbiochem), rabbit anti-Stim1 (Cell Signaling), anti-Bip/GRP78 (Abcam), anti-PARP (cleaved) (Cell Signaling), anti-Calsequestrin 2 (Thermo), anti-Fam20C (Tagliabracci et al., 2014 (link)), and anti-His (Invitrogen).

Pollak A.J., Liu C., Gudlur A., Mayfield J.E., Dalton N.D., Gu Y., Chen J., Heller Brown J., Hogan P.G., Wiley S.E., Peterson K.L, & Dixon J.E. (2018). A secretory pathway kinase regulates sarcoplasmic reticulum Ca2+ homeostasis and protects against heart failure. eLife, 7, e41378.

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Western Blot Analysis of Apoptosis Markers

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Equivalent amounts of proteins lysates were separated by 10% SDS-PAGE, transferred to 0.22μm PVDF membranes (Millipore, MA, USA), Membranes were blotted blocked in 5% fat-free milk in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature, followed by washing and incubated with anti-GATA3 (1:1000, Abcam), anti-Cleaved-PARP (1:1000, Cell Signaling Technology), anti-Cleaved-Caspase3 (1:1000, Abcam), STAT3(1:1000, Abcam), p-STAT3(1:1000, Abcam), anti-NF-κB (1:1000, Abcam) at 4°C overnight. Subsequently, the membranes were incubated with HRP-conjugated IgG for 2h at room temperature and detected with an enhanced chemiluminescence system. A GAPDH antibody was used as control. The experiment was performed in triplicate.

Zheng R., Jia J., Guan L., Yuan H., Liu K., Liu C., Ye W., Liao Y., Lin S, & Huang O. (2020). Long noncoding RNA lnc-LOC645166 promotes adriamycin resistance via NF-κB/GATA3 axis in breast cancer. Aging (Albany NY), 12(10), 8893-8912.

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Western Blot Analysis of Apoptosis Markers

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The following antibodies were used in western blot: monoclonal rabbit anti-Smac (Epitomics), rabbit polyclonal anti-GAPDH (Abcam), rabbit polyclonal anti-cleaved PARP, anti-cleaved caspase-9, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology), and rabbit polyclonal anti-VSV N (custom-made for the Luo lab). Details of antibody and caspase inhibitor usage are listed in Table 1.Table 1

Antibodies and Inhibitors

	Catalog No.	Vendor	Working Dilution
Inhibitors

Caspase-8 inhibitor (Z-IETD-FMK)	FMK007	R&D Systems	50 nM
Caspase-9 inhibitor (Z-LEHD-FMK)	FMK008	R&D Systems	50 nM

Reagents (Antibodies)

Anti-cIAP1	PA5-20066	Thermo Fisher	2 μg/mL
Anti-cIAP2	PA5-51700	Thermo Fisher	0.5 μg/mL
Anti-survivin	sc-17779	SCBT	1:300
anti-GAPDH	sc-32233	SCBT	1:1,000
anti-cleaved caspase-3	ab2302	Abcam	10 μg/mL
anti-cleaved PARP1 [4B5BD2]	ab110315	Abcam	1:1,000
Anti-VSV N	rabbit polyclonal	Luo lab	1:5,000

Li W., Turaga R.C., Li X., Sharma M., Enadi Z., Dunham Tompkins S.N., Hardy K.C., Mishra F., Tsao J., Liu Z.R., Fan D, & Luo M. (2019). Overexpression of Smac by an Armed Vesicular Stomatitis Virus Overcomes Tumor Resistance. Molecular Therapy Oncolytics, 14, 188-195.

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Evaluating Anticancer Drug Efficacy

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The cells were treated with ATF₂₄-PEG-Lipo-β-E and/or DDP for the indicated times. The PVDF membranes were incubated with anti-cyclin B1 (1:1,000, Cell Signaling Technology, anti-Bcl-2 (1:1,000; Cell Signaling Technology), anti-Bax (1:1,000; Cell Signaling Technology), anti-cleaved PARP (1:1,000; Cell Signaling Technology), anti-Cdc25C (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cdc2 p34 (1:200; Santa Cruz Biotechnology), anti-cleaved caspase-3 (1:400; Abcam, Cambridge, UK), and anti-GAPDH (1:1,000; Cell Signaling Technology) primary antibodies at 4 °C overnight. The other steps were the same as described above³⁸.

Zhai B., Chen P., Wang W., Liu S., Feng J., Duan T., Xiang Y., Zhang R., Zhang M., Han X., Chen X., Li Q., Li G., Liu Y., Huang X., Zhang W., Pan T., Yan L., Jin T., Xie T, & Sui X. (2020). An ATF24 peptide-functionalized β-elemene-nanostructured lipid carrier combined with cisplatin for bladder cancer treatment. Cancer Biology & Medicine, 17(3), 676-692.

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Immunoblotting Analysis of DNA Damage Response

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A2780, HEC1B and HeLa cells were plated and treated according to the following conditions and as described above for the cell viability or the clonogenic assays. Cells were washed with cold PBS and lysed 1% SDS, 150 mM NaCl, 5 mM NaF, 1% Tween 20, 0.5% NP40, 50 mM Tris–HCl pH 7.5 and 1× Halt protease and phosphatase inhibitor (Pierce). Equivalent amounts of protein lysates were resolved via 4–15% mini-PROTEIN TGX gels (Bio-Rad) and transferred to PVDF membranes. Membranes were blocked for 1 h using 5% non-fat dry milk (Bio-Rad) and incubated with primary antibody overnight at 4 °C, secondary antibody for 4 h at ambient temperature or overnight at 4 °C, and SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher Scientific) for 5 min. Anti-pATM (S1981) and anti-β actin were from Abcam. Anti-ATM was from Thermo Scientific. Anti-ATR, anti-pChk1 (S345), anti-Chk1, anti-pChk2 (Thr68), anti-Chk2, anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, goat anti-mouse and goat anti-rabbit IgG HRP-linked were from Cell Signaling Technologies. All primary antibodies were used at a 1:1000 dilution. Immunoblots were first probed for cleaved PARP followed by total PARP (Fig. 5B). Similarly, blots were first probed for cleaved caspase 3 followed by total caspase 3 (Fig. 5C and D). Images were acquired using a ChemiDoc XRS+ system (Bio-Rad).

Teng P.N., Bateman N.W., Darcy K.M., Hamilton C.A., Maxwell G.L., Bakkenist C.J, & Conrads T.P. (2015). Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells. Gynecologic oncology, 136(3), 554-561.

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Signaling Pathway Modulation in Cancer

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BMS-345541 was purchased from Sigma (Saint-Quentin-Fallavier, France). Vemurafenib, dabrafenib and trametinib were purchased from Euromedex (Souffelweyersheim, France) and TNFα was from PeproTech (Neuilly-Sur-Seine, France). Antibodies against CD271 was purchased from BD Biosciences (Franklin Lakes, NJ, USA), anti-ERK(D-2); anti-cleaved-PARP, anti-cleaved caspase-3, anti-pERK, anti-caspase-3, anti-JARID1B and TNFα blocking antibody were from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-IKK and anti-IκBα monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ABCB5 monoclonal antibody was purchased from Novus Biologicals (Abingdon, UK). Annexin V was purchased from Roche Diagnostic corporation (Indianapolis, IN, USA).

Lehraiki A., Cerezo M., Rouaud F., Abbe P., Allegra M., Kluza J., Marchetti P., Imbert V., Cheli Y., Bertolotto C., Ballotti R, & Rocchi S. (2015). Increased CD271 expression by the NF-kB pathway promotes melanoma cell survival and drives acquired resistance to BRAF inhibitor vemurafenib. Cell Discovery, 1, 15030-.

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Multimodal Cancer Treatment Investigation

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The 5-fluorouracil, metformin, and ICG-001 used were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA) respectively. The antibodies, such as anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, anti-LC3B, anti-ATG9A, anti-CD44, anti-β-catenin, anti-OCT4, anti-β-actin, anti-p-AMPK, and anti-AMPK were procured from Cell Signaling Technology (Danvers, MA, USA). APC conjugated anti-CD44 was procured from BioLegend (San Diego, CA, USA). All the primers were procured from Integrated DNA Technologies (San Diego, CA, USA).

Roy S., Zhao Y., Yuan Y.C, & Goel A. (2022). Metformin and ICG-001 Act Synergistically to Abrogate Cancer Stem Cells-Mediated Chemoresistance in Colorectal Cancer by Promoting Apoptosis and Autophagy. Cancers, 14(5), 1281.

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Western Blot Analysis of Cellular Signaling

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Protein aliquots of 25 μg each were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were washed three times and then incubated with Blocking One solution (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against anti-RET (D3D8R) (#14698), anti-phospho-RET (Tyr905) (#3221), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#4060), anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9664), and anti-β-actin (13E5) (#4970) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA). Additional antibodies were also used including anti-human/mouse/rat extracellular signal-regulated kinase (ERK) 1/ERK2 (0.2 μg/mL) (AF1576) and anti-phospho-ERK1/ERK2 (T202/Y204) (0.1 μg/mL) (AF1018) from R&D Systems. The membranes were washed three times and then incubated for 1 hour at room temperature with species-specific horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Each experiment was performed independently at least three times.

Arai S., Kita K., Tanimoto A., Takeuchi S., Fukuda K., Sato H, & Yano S. (2017). In vitro and in vivo anti-tumor activity of alectinib in tumor cells with NCOA4-RET. Oncotarget, 8(43), 73766-73773.

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