Anti mre11

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-MRE11 is a laboratory research antibody that recognizes the MRE11 protein. MRE11 is a critical component of the MRE11-RAD50-NBS1 (MRN) complex, which is involved in DNA double-strand break repair and telomere maintenance.

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MRE11 (31 citations)

15 protocols using anti mre11

Identification of rLOX-PP Interactors

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Empty vector and rLOX-PP expressing PC3 and DU145 cells were cultured, extracted into non-denaturing RIPA cell lysis buffer and incubated with agarose bead-immobilized Non-immune IgG or anti-c-Myc tag IgG overnight at 4°C to pull down rLOX-PP. Non-immune IgG (#31903, Thermo Scientific, Waltham, MA) was coupled to agarose using AminoLink Immobilization Plus Trial kit (#20394, Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. For pull-down assays, beads were washed, and then eluted according to kit instructions (ProFound c-Myc Tag IP/Co-IP Kit; Thermo Scientific, Waltham, MA). Eluted samples were analyzed by Western blotting on denaturing SDS PAGE probed with an anti-ATM, anti-ATR, Rad50, anti-NBS1 and anti-MRE11 antibodies (Cell Signaling, Danvers, MA) and with anti-rLOX-PP antibody (61 (link)). Aliquots (5%) of initial extracts taken before the immunoprecipitation were analyzed by Western blot on the same gels.

Bais M.V., Ozdener G.B., Sonenshein G.E, & Trackman P.C. (2014). Effects of Tumor Suppressor Lysyl Oxidase Propeptide on Prostate Cancer Xenograft Growth and Its Direct Interactions with DNA Repair Pathways. Oncogene, 34(15), 1928-1937.

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Western Blot Analysis of DNA Repair Proteins

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Cell lysates were obtained for validation of siRNA knockdown. Proteins were quantified using Bio-Rad protein assay (Bio-Rad). Proteins (30 μg per lane) were separated through a 4–15% (w/v) precast SDS polyacrylamide gel (Bio-Rad) and transferred onto PVDF membrane (Bio-Rad). Blots were immunostained with anti-MRE11 (rabbit, 1:5000, Cell Signaling), anti-NBS1 (rabbit, 1:5000, Cell Signaling), followed by HRP-conjugated secondary antibodies and ECL detection. Anti-β-actin monoclonal antibody (mouse, 1:10000, Santa Cruz Biotechnology) was used as protein loading control.

Sun Y., Soans E., Mishina M., Petricci E., Pommier Y., Nitiss K.C, & Nitiss J.L. (2022). Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells. Frontiers in Molecular Biosciences, 9, 1007064.

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Immunofluorescence Analysis of DNA Damage Markers

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Antibodies against TOP1, IκBα, phospho‐ATM, γH2AX, DNA. FLAG, β‐actin, Anti‐IKKγ/NEMO TDP1 (#SAB1411073), and H2AX were purchased from Sigma (St. Louis, MO). Antibodies against RPA2 and phospho‐RPA2 were from Bethyl Laboratories (Montgomery, TX). Antibodies against p65, WRN (#SC5629), ATM (#SC23921), CHK1 (#SC8404), Lamin B (#SC6216), and CRISPR‐Cas9 double nickase plasmid (control and WRN) were from Santa Cruz biotechnology (Santa Cruz, CA). Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK). Anti‐BrdU (#347580), anti‐PAR from BD Biosciences (San Jose, CA). Anti MRE11 (#4847) from Cell Signaling Technology (Massachusetts, USA). AlexaFlour‐546 and AlexaFlour‐488 tagged secondary antibodies from Jackson ImmunoResearch, (PA, USA). Lipofectamine 2000/3000, Alexa Flour‐488/555/595 (#A21123/#A31570), prolong anti‐fade Gold was obtained from (Life Technologies, Carlsbad, CA). SCH900776 (CHK1 inhibitor) was purchased from Selleckchem (Houston, USA). Talazoparib (BMN673; PARP inhibitor) was procured from ApexBio, USA. All other reagents like Ro106‐9920, mirin (MRE11 inhibitor), camptothecin, EdU, etc., were obtained from Sigma Chemicals (St. Louis, MO), unless mentioned in the respective places.

Gupta P., Majumdar A.G, & Patro B.S. (2022). Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer. Aging Cell, 21(6), e13625.

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Antibodies for Immune Signaling Analysis

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The primary antibodies anti-CARD9 (sc-99054), anti-Rad50 (sc-56209 and sc-74460), anti-caspase-1 p10 (sc-514), anti-Lamin B (sc-6217), anti-NF-κB p65 (sc-372), and anti-cRel (sc-71) were obtained from Santa Cruz, and anti-Bcl10 (#4237), anti-Mre11 (#4895), anti-p95/Nbs1 (#3002), anti-IRF-3 (#4302), and anti-phospho-IRF-3 (Ser396) (#4947) antibodies were obtained from Cell Signaling. anti-CARD9 (a rabbit polyclonal antibody raised against an N-terminal peptide of CARD9) was kindly provided by Margot Thome. The secondary anti-mouse (donkey) and anti-rabbit antibodies (goat) for immunofluorescence were conjugated to Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes), respectively.

Roth S., Rottach A., Lotz-Havla A.S., Laux V., Muschaweckh A., Gersting S.W., Muntau A.C., Hopfner K.P., Jin L., Vanness K., Petrini J.H., Drexler I., Leonhardt H, & Ruland J. (2014). Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production. Nature immunology, 15(6), 538-545.

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Western Blot Analysis of DNA Damage Response

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Proteins (20 μg) were loaded and separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels at 15 mA for 60 min. Then, the separated proteins were transferred to 0.45 µm Amersham Protran Nitrocellulose Blotting Membrane (GE Healthcare, Life Sciences, Marlborough, MA, USA) in methanol transfer buffer using Mini Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing Tween 20 (TBST; 20 mM of Tris–HCl, pH 7.4, 0.15 M of NaCl, and 0.1% Tween 20) for 1 h and incubated with anti-MRE11, anti-γH2AX, anti-RAD51 (Cell Signaling, Leiden, the Netherlands) and anti-GAPDH antibodies (Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Abcam, Cambridge, UK). The membranes were then incubated with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore Corporation, Billerica, MA, USA) and visualized by Azure c600 (Azure Biosystems, Dublin, CA, USA).

Horak J., Dolnikova A., Cumaogullari O., Cumova A., Navvabi N., Vodickova L., Levy M., Schneiderova M., Liska V., Andera L., Vodicka P, & Opattova A. (2022). MiR-140 leads to MRE11 downregulation and ameliorates oxaliplatin treatment and therapy response in colorectal cancer patients. Frontiers in Oncology, 12, 959407.

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Western Blot Analysis of DNA Repair Proteins

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Cells were collected by trypsinization and lysed in RIPA lysis buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris base, pH 8.0). Phosphatase and protease inhibitors (Gold Biotechnology) were added freshly to the lysis buffer. Following gel electrophoresis and transfer of cell extracts onto nitrocellulose, membranes were incubated for 1 hr or overnight in blocking buffer (5% milk in TBS + 0.1% tween). Membranes were subsequently incubated with primary antibodies diluted in antibody buffer (3% BSA in TBS + 0.1% tween) for 2 hrs at room temperature or overnight at 4°C. Detection was achieved using appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. Anti-ZRANB3 (1:5,000, Bethyl Laboratories), anti-SMARCAL1 (1:1,000, Santa Cruz Biotechnology), anti-BRCA1 (1:500, Bethyl Laboratories), anti-BRCA2 (1:1,000, Bethyl Laboratories), anti-vinculin (1:100,000, Sigma-Aldrich), anti-β-actin (1:100,000, Novus Biologicals), anti-GAPDH (1:5,000, Novus Biologicals), anti-HLTF (1:2,000, Abcam), anti-LAMIN B1 (Thermo Fisher Scientific), anti-FANCD2 (1:1,000, Novus Biologicals), anti-PCNA (1:100,000, Abcam), anti-NBS1 (GeneTex), anti-MRE11 (Cell Signaling Technology), anti-RPA2 (Bethyl Laboratories) antibodies were used in western blot experiments.

Taglialatela A., Alvarez S., Leuzzi G., Sannino V., Ranjha L., Huang J.W., Madubata C., Anand R., Levy B., Rabadan R., Cejka P., Costanzo V, & Ciccia A. (2017). Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers. Molecular cell, 68(2), 414-430.e8.

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Molecular Targets in Cancer Research

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Anti-NF-κB, anti-Bcl-2, anti-cyclin A, anti-cyclin B, anti-cyclin E, anti-phospho-Raf-1, anti-Akt, anti-Snail, and anti-β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-cleaved PARP, caspase-3, anti-ATM, anti-ATR, anti-Rad50, anti-p95/NBS1, anti-Rad52, anti-MRE11, anti-Ku70, anti-Ku80, anti-DNA-PKcs, anti-ERCC1, anti-Rad51, anti-Ras, anti-MEK1/2, anti-phospho-MEK1/2, anti-Erk1/2, anti-phospho-Erk1/2, anti-phospho-Akt, anti-vimentin, anti-E-cadherin, and anti-MMP9 were purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-γ-H2AX was obtained from Millipore (Billerica, MA, USA). ZOL was purchased from Sigma-Aldrich (St. Louis, MO, USA). For in vitro experiments, ZOL was dissolved in PBS to make a 2 mmol/L stock solution and stored at −20°C.

Kim E.H., Kim M.S., Lee K.H., Koh J.S., Jung W.G, & Kong C.B. (2016). Zoledronic acid is an effective radiosensitizer in the treatment of osteosarcoma. Oncotarget, 7(43), 70869-70880.

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Western Blot Analysis of DNA Damage Response Proteins

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The antibodies from this study were as follows: anti-keratin-10, anti-loricrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA); anti-E2F1, anti-p63, anti-ATR, anti-p-ATR, anti-CHK1, anti-p-CHK1, anti-CHK2, anti-p-CHK2, anti-RAD51, anti-Mre11, anti-NBS1, anti-BRCA1, and anti-BRCA2 (Cell Signaling, Danvers, MA); anti-p73 (Abcam, Cambridge, MA); anti-p53 (Millipore, Burlington, MA); anti-p-TopBP1 (Abgent, San Diego, CA). AKT inhibitors MK2206 (2μM) and LY294002 (25μM) were purchased from SelleckChem (Houston, TX). Cell lysates were processed and assayed by western blot analysis as previously described ^{60 (link)}. Briefly, J2 feeders were firstly removed by Versene (PBS containing 0.5 mM EDTA) treatment and keratinocytes were collected and lysed in RIPA lysis buffer on ice for 30 minutes. The samples were then electrophoresed on SDS-page gels and transferred to PVDF membranes. At last the membranes were developed using ECL prime or ECL reagents (Amersham, Pittsburgh, PA) and chemiluminescence signals were detected using Eastman Kodak x-ray films.

Hong S., Xu J., Li Y., Andrade J., Hoover P., Kaminski P.J, & Laimins L.A. (2019). Topoisomerase IIβ-binding protein 1 activates expression of E2F1 and p73 in HPV positive cells for genome amplification upon epithelial differentiation. Oncogene, 38(17), 3274-3287.

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Chromatin Protein Fraction Isolation

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The chromatin-bound protein fraction was isolated as described [24 (link)-26 (link)] with minor modifications. Western blots were performed with mouse monoclonal anti-γH2AX (JBW301; 1:4000), rabbit polyclonal anti-HsRAD51 (H92, Santa Cruz Biotechnology; 1:1000), rabbit polyclonal anti-Mre11 (Cell Signaling Technology; 1:500) and rabbit polyclonal anti-H3 (C-16, Santa Cruz Biotechnology; 1:3000).

Kim T.M., Son M.Y., Dodds S., Hu L, & Hasty P. (2014). Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks. Mutation research, 766-767, 66-72.

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Antibody Characterization and Validation

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Commercial antibodies used were as follows: anti-FLAG (Sigma), anti-CDYL (Sigma, HPA035578; for Western blotting), anti-CDYL (Abcam, ab5188), anti-BrdU (Abcam, ab6326), anti-RPA1 (ABclonal, A0990), anti-RAD51 (ABclonal, A6268), anti-WRN (ABclonal, A6855), anti-DNA2L (Abcam, ab2179), anti-BLM (Abcam, ab96488), anti-MRE11 (Cell Signaling Technology, 4847), anti-NBS1 (Cell Signaling Technology, 14956), anti-RAD50 (Cell Signaling Technology, 3427), anti-Myc, anti-hemagglutinin (HA), anti–β-actin (MBL), anti-PanKac (PTM, 101), anti-PanKcr (PTM, 502; for Western blotting), and anti-PanKcr–conjugated agarose beads (PTM, 503; for immunoprecipitation). A CDYL antibody that we generated previously (peptide antigen: KQKESTLTRTNRTSPNN; B&M) was used for immunoprecipitation. The polyclonal antibodies against RPA1 K88cr, RPA1 K379cr, and RPA1 K595cr were generated by immunizing rabbits with two synthetic crotonyl peptides corresponding to residues surrounding K88, K379, and K595 of RPA1, respectively. HU, CPT, and VP16 were from Sigma. Streptavidin agarose resins were from Thermo Fisher Scientific. Protein A/G and Sepharose CL-4B beads were from GE Healthcare Biosciences, and protease inhibitor mixture cocktail was from Roche Applied Science.

Yu H., Bu C., Liu Y., Gong T., Liu X., Liu S., Peng X., Zhang W., Peng Y., Yang J., He L., Zhang Y., Yi X., Yang X., Sun L., Shang Y., Cheng Z, & Liang J. (2020). Global crotonylome reveals CDYL-regulated RPA1 crotonylation in homologous recombination–mediated DNA repair. Science Advances, 6(11), eaay4697.

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