- 436400 - Invitrogen Antibodies PARP1 antibody

Submitted references Infigratinib (BGJ 398), a Pan-FGFR Inhibitor, Targets P-Glycoprotein and Increases Chemotherapeutic-Induced Mortality of Multidrug-Resistant Tumor Cells.

Boichuk S, Dunaev P, Mustafin I, Mani S, Syuzov K, Valeeva E, Bikinieva F, Galembikova A
Biomedicines 2022 Mar 3;10(3)

Modulation of Epithelial Mesenchymal Transition after AGTR-1 Gene Edition by Crispr/Cas9 and Losartan Treatment in Mammary Tumor Cell Line: A Comparative Study between Human and Canine Species.

Moschetta-Pinheiro MG, Colombo J, Godoy BLV, Balan JF, Nascimento BC, Zuccari DAPC
Life (Basel, Switzerland) 2021 Dec 18;11(12)

Inhibition of AKT-Signaling Sensitizes Soft Tissue Sarcomas (STS) and Gastrointestinal Stromal Tumors (GIST) to Doxorubicin via Targeting of Homology-Mediated DNA Repair.

Boichuk S, Bikinieva F, Nurgatina I, Dunaev P, Valeeva E, Aukhadieva A, Sabirov A, Galembikova A
International journal of molecular sciences 2020 Nov 22;21(22)

Proteomics Profiling of KAIMRC1 in Comparison to MDA-MB231 and MCF-7.

Alghanem B, Ali R, Nehdi A, Al Zahrani H, Altolayyan A, Shaibah H, Baz O, Alhallaj A, Moresco JJ, Diedrich JK, Yates JR 3rd, Boudjelal M
International journal of molecular sciences 2020 Jun 18;21(12)

Establishment and characterization of a triple negative basal-like breast cancer cell line with multi-drug resistance.

Boichuk S, Galembikova A, Sitenkov A, Khusnutdinov R, Dunaev P, Valeeva E, Usolova N
Oncology letters 2017 Oct;14(4):5039-5045

USP17-mediated deubiquitination and stabilization of HDAC2 in cigarette smoke extract-induced inflammation.

Song H, Tao L, Chen C, Pan L, Hao J, Ni Y, Li D, Li B, Shi G
International journal of clinical and experimental pathology 2015;8(9):10707-15

TIMELESS Forms a Complex with PARP1 Distinct from Its Complex with TIPIN and Plays a Role in the DNA Damage Response.

Young LM, Marzio A, Perez-Duran P, Reid DA, Meredith DN, Roberti D, Star A, Rothenberg E, Ueberheide B, Pagano M
Cell reports 2015 Oct 20;13(3):451-459

The Deubiquitinase USP17 Regulates the Stability and Nuclear Function of IL-33.

Ni Y, Tao L, Chen C, Song H, Li Z, Gao Y, Nie J, Piccioni M, Shi G, Li B
International journal of molecular sciences 2015 Nov 24;16(11):27956-66

Deubiquitination and stabilization of IL-33 by USP21.

Tao L, Chen C, Song H, Piccioni M, Shi G, Li B
International journal of clinical and experimental pathology 2014;7(8):4930-7

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of PARP was achieved by transfecting HeLa cells with PARP specific validated siRNA (Silencer® select Product # s1097). Western blot analysis (Fig a) was performed using whole cell lysates from the PARP knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-PARP Mouse Monoclonal Antibody (Product # 436400, 1-2 µg/mL) and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to PARP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Jurkat (lane 1), Jurkat treated for Overnight with 1 uM of Etoposide (lane 2), HeLa (lane 3), HeLa treated for Overnight with 1 uM of Etoposide (lane 4) HeLa treated for Overnight with 3 uM of Staurosporine (lane 5) and MDA-MB-231 (lane 6). The blots were probed with Anti-PARP Mouse Monoclonal Antibody (Product # 436400, 1-3 µg/mL) and detected by chemiluminescence Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). Along with 116 kDa, 89 kDa Caspase cleaved isoform band corresponding to PARP was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing of PARP1 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR978664_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of PARP1 was performed by loading 30 µg of HeLa CAS9 (Lane 1) and HeLa CAS9 cells transduced with PARP1 Lentiviral sgRNA (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ 3 to 8%, Tris-Acetate, 1.0 mm, Mini Protein Gel (Product # EC66952BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-PARP1 Monoclonal Antibody (123)(Product # 436400) using 1 µg/mL dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific to PARP1 (Fig (b)). Cleaved PARP1 bands were observed in HeLa CAS9 (Lane 1) and HeLa CAS9 cells transduced with PARP1 Lentiviral sgRNA (Lane 2) at ~80 kDa. Uncharacterized band was observed in HeLa Cas9 (Lane 1) at ~60 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PARP was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with PARP Mouse Monoclonal Antibody (Product # 436400) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing both nuclear and cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of PARP showing staining in the nucleus of paraffin-embedded human astroglioma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PARP Mouse Monoclonal Antibody (Product # 436400) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of PARP showing staining in the nucleus of paraffin-embedded human diffuse large B cell lymphoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PARP Mouse Monoclonal Antibody (Product # 436400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometric analysis of Hela cells, green: 0 µg/mL of primary antibody, red: 2.5 µg/mL of primary antibody using IgG (H+L), Goat F (ab´)2 Anti-Mouse, (R-PE) (Product # M350043) secondary antibody, and Product # GAS-003 for Fix and Perm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 TIMELESS Physically Interacts with PARP1 (A) HEK293T cells were transfected with the indicated FLAG-tagged proteins. 24 hr post-transfection, cells were harvested and lysed. Cell extracts were subjected to immunoprecipitation with alpha-FLAG resin and stained with Ponceau S. MWM, molecular weight markers. (B) The experiment was performed as in (A), except that whole-cell extracts (WCEs) were subjected to immunoprecipitation (IP) with alpha-FLAG resin and immunoblotted as indicated. EV, empty vector. Asterisks denote FLAG-tagged proteins. (C) The experiment was performed as in (B). (D) Lysates of U2OS cells were immunoprecipitated with either an antibody against TIMELESS or a rabbit IgG and immunoblotted as indicated. (E) Super-resolution images of TIMELESS and PARP1 in U2OS cells selected from regions of the nucleus shown in Figure S1D . Scale bar, 400 nm. (F) Super-resolution images of TUNEL, TIMELESS, and PARP1 in neocarzinostatin treated U2OS cells showing that TIMELESS and PARP1 are found at DNA damage sites.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Identification of the PARP1 Binding Site in TIMELESS (A) HEK293T cells were transfected with either an empty vector (EV), FLAG-tagged TIMELESS, or FLAG-tagged TIMELESS mutants as indicated. Asterisks indicate the insertion of a STOP codon after the numerically specified codon. 24 hr post-transfection, WCEs were subjected to IP with alpha-FLAG resin and immunoblotted as indicated. (B) Schematic representation of TIMELESS mutants tested for binding to PARP1. TIMELESS mutants that interacted with endogenous PARP1 are designated with the symbol (+); those unable to co-precipitate PARP1 are designated with the symbol (-). (C) The experiment was performed as in (A), except that different TIMELESS mutants were used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: RNA immunoprecipitation (RIP) western of PARP was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of PARP monoclonal antibody (Product # 436400) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a PARP monoclonal antibody (Product # 436400) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of PARP1. Cells were seeded onto a 10 cm plate and allowed to grow overnight in DMEM/F12 + 10% FBS at 37°C to achieve ~80% confluence. Media was changed and menadione in ethanol was added to a final concentration of 100 µM. Cells were incubated at 37°C for 20 minutes then washed once in 1xPBS and moved to 4°C. Cells were collected in 500 µl PBS with 0.5% triton X-100 and 0.5 mM PMSF. Cells were sonicated on ice, sample was spun at 15,000 rpm at 4°C for 5 minutes and supernatant was collected. 30 µl was collected for input and 2x80 µl was collected for antibody vs IgG control (Bead). 1 µl monoclonal PARP1 antibody (Product # 436400) was added to rotated overnight at 4°C (no antibody added to bead lanes). The next day, 5 µl protein G beads in BSA was added to the antibody and IgG control tubes and rotated at 4°C for 1.5 hours. After incubation, four washes in ice cold 1xPBS with 0.5% triton X-100 were performed. Finally, 20 µl 1xSDS sample buffer was added to IP/bead and 10 µl 4xSDS sample buffer was added to input and all samples were heated to 95°C for 5 minutes. 10 µl of each sample with input diluted 1:5 were run on a 4-12% Bis-Tris gel and probed with rbPARP1 antibody. Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Expression of MTAP, PARP1, KCTD12, and Lamin A/C in cancer cell lines: Western blot analysis of protein expression of MTAP, PARP1, KCTD12, Lamin A/C, and phosphorylated Lamin A/C in MDA MB231, MCF-7, and KAIMRC1 cells. Preparation of cell lysates and western blot were performed as described under the Materials and Methods section. In both normal (+) and serum-starved (-) conditions, KAIMRC1 cells showed strong expression of MTAP, PARP1, and KCTD12 compared to MDA-MB231 and MCF-7 cells. Moreover, KAIMRC1 cells showed weak expression of Lamin A/C and phosphorylated Lamin A/C in comparison to MDA MB231 cells, thus validating our proteomics results.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 AKT inhibition potentiates pro-apoptotic activity of Dox in STS and GIST. Immunoblot analysis for apoptosis markers (cleaved forms of poly-(ADP)-ribose-polymerase (PARP) and caspase-3) in RD rhabdomyosarcoma ( A ), U2-OS osteosarcoma ( B ), HT-1080 fibrosarcoma ( C ), GIST T-1R ( D ), GIST 430 ( E ) and SK-LMS-1 leiomyosarcoma ( F ) cells after treatment with DMSO (control), Dox (0.25 g/mL) and MK-2206 (5 M) alone and in combination (e.g., Dox + MK-2206) for 72 h. The lysates were also probed for total and phosphorylated forms of AKT to illustrate AKT inhibition by MK-2206. pAKT expression is shown by arrows. Actin was used as a loading control.

436400

Antibody data