Cleaved parp 5625

Manufactured by Cell Signaling Technology

Sourced in United States

Cleaved PARP (5625) is a monoclonal antibody that specifically recognizes the 89 kDa cleaved fragment of PARP. PARP (Poly(ADP-ribose) Polymerase) is a key enzyme involved in DNA repair. Cleavage of PARP by caspases is a hallmark of apoptosis.

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Lab products found in correlation

Cleaved caspase 3 9661, by Cell Signaling Technology (3 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) β actin a5441, by Merck Group (2 mentions) Rpmi 10 040 cm, by Corning (2 mentions) Nitrocellulose membranes 9004700, by Bio-Rad (2 mentions) Vinculin v9131, by Merck Group (2 mentions) Cleaved caspase 3 9664, by Cell Signaling Technology (2 mentions) Eif2α 9722, by Cell Signaling Technology (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Propidium iodide, by BD (1 mentions) N acetyl cysteine (nac), by Merck Group (1 mentions) Bcl2 sc 492, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit 1, by BD (1 mentions) Ros probe 2 7 dichlorodihydro fluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Muse mitopotential kit, by Merck Group (1 mentions) Pierce ecl western blotting detection kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Cleaved PARP (928 citations)

15 protocols using cleaved parp 5625

Apoptosis Assay Protocol with Inhibitors

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CHE was obtained from Aladdin (Shanghai, China). Antibodies against B-cell lymphoma 2 (Bcl2, sc-492), Bcl2-associated protein x (Bax, sc-493), GAPDH (sc-32233), and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cleaved form of poly (ADP-ribose) polymerase (cleaved-PARP, 5625S), phosphorylated eukaryotic initiation factor 2α (p-eIF2α, 3398S), eIF2α (9722S), and activating transcription factor-4 (ATF4, 11815S) were obtained from Cell Signaling Technology (Danvers, MA, USA). NAC, DMSO, and MTT were purchased from Sigma-Aldrich (St Louis, MO, USA). Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I and propidium iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). ROS probe 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).

Wu S., Yang Y., Li F., Huang L., Han Z., Wang G., Yu H, & Li H. (2018). Chelerythrine induced cell death through ROS-dependent ER stress in human prostate cancer cells. OncoTargets and therapy, 11, 2593-2601.

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Molecular Mechanisms of Apoptosis Regulation

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4-Aminopyridine (4-AP) and MTT were products of the Sigma Chemical Corp. (St. Louis, MO). Roswell Park Memorial Institute-1640 (RPMI-1640) medium, DMEM, and fetal bovine serum (FBS) were obtained from Life Technologies (Carlsbad, CA). Muse Annexin V and the Dead Cell Kit (MCH100105) and the Muse MitoPotential Kit (MCH100110) were procured from Millipore Corporation (Darmstadt, Germany). The caspase-3 activity assay kit (C1116), caspase-9 activity assay kit (C1158), and mycoplasma Stain Assay Kit (C0296) were obtained from Beyotime Institute of Biotechnology (Haimen, China). Protein extraction buffer (AR0105), protease (AR1182), and phosphatase inhibitors (AR1183) were obtained from Wuhan Boster Biological Engineering Co., Ltd (Wuhan, China). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), Apaf-1 (8969S), cleaved caspase-3 (9664S), cleaved caspase-9 (7237S), and cleaved PARP (5625S) were purchased from Cell Signaling Technology (Beverly, MA), and the antibodies were validated by the manufacturer. The miR-10-5p mimic, inhibitor, and negative control; pmiR-RB-Report control plasmid; pmiR-RB-Repor-Apaf-1-3′UTR wild-type plasmid; and mutant reporter plasmid were synthesized and purified by Guangzhou RiboBio Co., Ltd (Guangzhou, China). All other chemicals were of standard analytical grade.

Ru Q., Li W.L., Xiong Q., Chen L., Tian X, & Li C.Y. (2018). Voltage-gated potassium channel blocker 4-aminopyridine induces glioma cell apoptosis by reducing expression of microRNA-10b-5p. Molecular Biology of the Cell, 29(9), 1125-1136.

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Western Blot Analysis of Cell Signaling

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Following cell treatments, the whole cell lysates were collected after mixing cell pellets with 100 μL of radio immune precipitation assay buffer (RIPA). Protein sample concentration was determined by using the bicinchoninic acid protein assay kit (Pierce; USA). Each sample (20 μg) was electrophoresed through a 4–12% sodium dodecyl (SDS)-polyacrylamide gel (Life Technologies, UK), transferred onto a nitrocellulose membrane (Hybond-C, Amersham; UK), and probed with antibodies which include CD45 (PA5-95,187), JAK2 (MA5-15,632), ACTR2 (A305-216A-M), THAP3 (PA5-50,841), PBX-1 (PA5-29,674), and SEG (PA5-51,462), from ThermoFisher Scientific, UK. Other apoptotic antibodies such as caspase 3 (9662S), caspase 9 (9502S), cleaved caspase 9 (7237S), PARP (9542S), and cleaved PARP (5625S) were purchased from Cell Signaling, UK. Rabbit anti-rat HRP (ab6734) and rabbit anti-mouse HRP (58802S) antibodies were purchased from Abcam-UK. CRISPR-Cas9 (NBP2-36,440) was purchased from Novus. GAPDH was used as a loading control (Sigma; UK). Levels of protein expression were assessed using Pierce ECL western blotting detection kit (Thermo Scientific; UK). Membranes were scanned using benchtop G:box (Syngene; UK) and density was calculated using the image J program (http://rsbweb.nih.gov/ij/) incorporating correction of loading controls.

Al Barashdi M.A., Ali A., McMullin M.F, & Mills K. (2023). CD45 inhibition in myeloid leukaemia cells sensitizes cellular responsiveness to chemotherapy. Annals of Hematology, 103(1), 73-88.

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Antibody Panel for Signaling Pathways

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The following antibodies were purchased: pALK^Y1604 (Y664 in NPM-ALK; 3341), pALK^Y1586 (Y646 in NPM-ALK; 3343), pIGF-IR^Y1131 (3021), pSTAT3^Y705 (4113), AKT (9272), pAKT^S473 (4051), JNK (3708S), pJNK^T183/Y185 (4668S), PARP (9542P), cleaved PARP (5625S), and cleaved caspase-3 (9661S) (Cell Signaling Technology, Danvers, MA); pIGF-IR^Y1161 (Ab39398) (Abcam, Cambridge, MA); IGF-IR (39-6700) (Invitrogen, Grand Island, NY); STAT3 (sc-8019) and caspase-3 (sc-7272) (Santa Cruz Biotechnology, Santa Cruz, CA); ALK (M719501-2) and Ki-67 (M7240) (Dako, Carpinteria, CA); and β-actin (A-2228) (Sigma-Aldrich, St. Louis, MO).

George S.K., Vishwamitra D., Manshouri R., Shi P, & Amin H.M. (2014). The ALK inhibitor ASP3026 eradicates NPM-ALK+ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model. Oncotarget, 5(14), 5750-5763.

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Western Blot Analysis of Apoptotic Markers

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Whole cell lysates were prepared as described previously.30 (link) Protein concentration was determined using Pierce^TM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Cell lysates (50 µg) were electrophoresed through 4–20% precast polyacrylamide gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes (GE healthcare, USA). The blots were blocked in Tris-Buffered Saline and Tween 20 (TBST) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) containing 4% milk and probed with the antibody GADD45α (H-165), β-actin (C4) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), caspase-3 (9662), cleaved caspase-3 (9661) or cleaved PARP (5625) (Cell Signaling Technology, MA, USA) diluted according to manufacturer’s recommendation in TBST with 5% Bovine Serum Albumin (Boston BioProducts, Ashland, MA, USA) plus 0.02% sodium azide (NaN₃) overnight at 4ºC. After washed with TBST, the blots were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit antibody for 1 hr, and then washed and detected by the West Pico from SuperSignal (Thermo Scientific, Waltham, MA, USA).

Shi Q., Sutariya V., Varghese Gupta S, & Bhatia D. (2019). GADD45α-targeted suicide gene therapy driven by synthetic CArG promoter E9NS sensitizes NSCLC cells to cisplatin, resveratrol, and radiation regardless of p53 status. OncoTargets and therapy, 12, 3161-3170.

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Lung Cancer Cell Line Investigation

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The human lung cancer cell lines NCI-H1299 (ATCC® CRL-5803™) and NCI-H1975 (ATCC® CRL-5908™) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere. All reagents for the cell culture were purchased from Gibco Life Technologies (Grand Island, NY, USA), unless otherwise stated.
Tubeimoside I (Tub, T2715) was purchased from Targetmol (Boston, MA, USA). Bafilomycin A1 (Baf, S1413), acetylcysteine (NAC, S1623) and rapamycin (Rapa, S1039) were purchased from Selleckchem (Houston, TX, USA). Primary antibodies against β-actin (3700), LC3B (3868), p62 (88588), cathepsin B (31718), cathepsin D (2284), Caspase 3 (9662), p-DRP1 (ser616) (3455), p-DRP1 (ser637, (4867), cytochrome C (11940), COX 4 (4850), and cleaved-PARP (5625) were obtained from Cell Signaling Technology (Boston, MA, USA). Cathepsin L (AF952) was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Anti-Bax (6A7) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, United States). The secondary antibodies peroxidase-labeled anti-mouse IgG (AS004) and anti-rabbit IgG (AS014) were obtained from Abclonal (Wuhan, China).

Wang K., Zhan Y., Chen B., Lu Y., Yin T., Zhou S., Zhang W., Liu X., Du B., Wei X, & Xiao J. (2020). Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria. Cell Death & Disease, 11(8), 708.

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Western Blot Analysis of DNA Damage Response

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Cells were scraped in PBS with 2 mmol/L Na₃VO₄ and cell pellets lysed in 50 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mmol/L Na₃VO₄, and protease inhibitors. In vivo samples were processed by homogenization using a Precellys24 homogenizer (Bertin Technologies). Protein supernatants, quantified by the BCA assay (Pierce), were separated by SDS-PAGE, transferred to PVDF membrane (Thermo Scientific) and blocked in TBS with 5% non-fat dry milk and 0.1% Tween-20. Membranes were probed using the following antibodies: phospho-ATM Ser1981, #5883; ATM, #2873; p21 #2946; phospho-p53 Ser15, #2528; GAPDH, #2118; Chk1, #2360; phospho-Chk1 Ser345, #2341; Chk2, #2662; phospho-Chk2 Thr68, #2197; γH2AX, #9718; cleaved caspase 3, #9661 and cleaved PARP #5625, were all obtained from Cell Signaling Technology. p53 DO-7 mAb #M7001 was obtained from Dako and PARP-1 from Santa Cruz Biotechnology. For confocal microscopy on fixed cells, γH2AX JBW-301 (Merck-Millipore) and Rad51 H-92 (Santa Cruz 8349) were used.

Dillon M.T., Barker H.E., Pedersen M., Hafsi H., Bhide S.A., Newbold K.L., Nutting C.M., McLaughlin M, & Harrington K.J. (2016). Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric Micronuclei. Molecular cancer therapeutics, 16(1), 25-34.

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Immunoblotting of Apoptosis and Redox Markers

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Cells were subjected to drug treatment at a density of 0.1–0.2 mln/ml. Cell lysis, protein concentration measurement and immunoblotting was performed as described in [21 (link)]. Antibodies used: Cell Signalling: cleaved PARP - 5625, caspase 3 - 9662, p21 Waf1/Cip1 – 2947, biotin HRP-linked – 7075, Akt1 - 2967, phospho-Akt (Thr308) – 2965, phospho-p44/42 MAPK (P-ERK1/2) (Thr202/Tyr204) – 9101, p44/42 MAPK (ERK1/2) – 9107, TRX1 - 2429; Sigma-Aldrich: PRDX1 - HPA007730, PRDX6 - HPA006983, β-actin-HRP – A3854; Abcam: PRDX2 - EPR5155; AbFrontier: PRDX3 - LF-MA0044, PRDX4 - LF-MA0014, PRDX5 - LF-MA0002, Life Technologies: V5 - R96025; Santa Cruz Biotechnology Inc.: TRXR1 – sc-20147, Enzo Life Sciences: CRT - ADI-SPA-600.

Trzeciecka A., Klossowski S., Bajor M., Zagozdzon R., Gaj P., Muchowicz A., Malinowska A., Czerwoniec A., Barankiewicz J., Domagala A., Chlebowska J., Prochorec-Sobieszek M., Winiarska M., Ostaszewski R., Gwizdalska I., Golab J., Nowis D, & Firczuk M. (2015). Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma. Oncotarget, 7(2), 1717-1731.

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Immunoblotting Analysis of Cellular Signaling

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Human cell lines were cultured in RPMI (10-040-CM; Cellgro) supplemented with 5% fetal bovine serum and harvested at 70% confluence. For immunoblotting, cells were treated for the specified times with the indicated drugs, washed with cold phosphate buffered saline (PBS) containing 100 mM Na₃VO₄, and lysed using TNE buffer (150 mM NaCl, 1% (v/v) NP-40, 2 mM EDTA, 50 mM Tris-HCl, pH 8.0) supplemented with protease inhibitors (11697498001; Roche). Proteins were separated by SDS−PAGE and transferred to nitrocellulose membranes (9004700; BioRad). After blocking for 1 h in 5% (wt/vol) dry milk/Tris-buffered saline (TBS)/0.1% (v/v) Tween-20, membranes were incubated overnight at 4 °C with primary antibodies followed by incubation with Alexa Fluor-labeled secondary antibodies (IRDye 680LT goat-anti-mouse or IRDye 800CW goat-anti-rabbit antibodies (LI-COR Biosciences) for 1 h. β-Actin (A5441) and vinculin (V9131) antibodies were obtained from Sigma. p-AKT (4056, 4060), S6 (2317), p-S6 (4858, 5364), S6K1 (2708), p-S6K1 (9234), p-eEF2k (3691), peIF4B (3591), and cleaved PARP (5625) were obtained from Cell Signaling Technologies. Fluorescent images were acquired and by LI-COR Odyssey Imaging System.

Qin J., Rajaratnam R., Feng L., Salami J., Barber-Rotenberg J.S., Domsic J., Reyes-Uribe P., Liu H., Dang W., Berger S.L., Villanueva J., Meggers E, & Marmorstein R. (2014). Development of Organometallic S6K1 Inhibitors. Journal of Medicinal Chemistry, 58(1), 305-314.

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Cell lysis and Western blot analysis

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After treatments, cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₄P₂O₇, 1 mM β-glycerophosphate, 1 mM Na₃VO₄) supplemented with protease (Fisher Scientific, Pittsburgh, PA, #P1-88266) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, #04906845001), and Western blot was performed as previously described [44 (link)]. Membranes were incubated with primary antibodies pRb T821/826 #271930 (1:200), Rb #73598 (1:200), Cleaved Caspase 3 #9661 (1:1,000), Cleaved PARP #5625 (1:1,000), c-Myc #5605 (1:1,000), MCL-1 #4572 (1:1,000) (Cell Signaling Technology, Danvers, MA), VHL #564183 (1:500) (BD Biosciences, San Jose, CA), HIF-2α #NB100122 (1:1,000, Novus Biologicals, Littleton, CO), and β-actin #A5441 (1:5,000, Sigma Aldrich, St. Louis, MO) in 5% BSA/PBS/0.05%Tween20 (PBS-T) overnight at 4°C, and secondary antibody incubation with Goat anti-Rabbit IgG #31460 (1:5,000) (Thermo Fisher Scientific, Waltham, MA) and Goat anti-Mouse IgG #31430 (1:2,000) (Thermo Fisher Scientific, Waltham, MA) in 5% nonfat milk/PBS-T blocking buffer for 1 hour at room temperature (RT). The HRP signal was developed using ECL WB substrate (GE Healthcare, Scottsdale, AZ, USA; #RPN2236). Images were acquired on the Bio-Rad ChemiDoc XRS⁺ imaging system (BioRad, Hercules, CA).

Nelson L.J., Castro K.E., Xu B., Li J., Dinh N.B., Thompson J.M., Woytash J., Kipp K.R, & Razorenova O.V. (2022). Synthetic lethality of cyclin-dependent kinase inhibitor Dinaciclib with VHL-deficiency allows for selective targeting of clear cell renal cell carcinoma. Cell Cycle, 21(10), 1103-1119.

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