Mre11

Manufactured by Abcam

Sourced in United States

MRE11 is a core component of the MRE11-RAD50-NBS1 (MRN) complex, which is involved in the recognition and repair of DNA double-strand breaks. MRE11 possesses both exonuclease and endonuclease activities, playing a crucial role in the initial processing of DNA ends during the DNA damage response.

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Lab products found in correlation

Gapdh, by Abcam (2 mentions) γh2ax, by Cell Signaling Technology (1 mentions) Flag tag (flag), by Santa Cruz Biotechnology (1 mentions) Alexa fluor 680, by Thermo Fisher Scientific (1 mentions) Rpa32, by Santa Cruz Biotechnology (1 mentions) Samhd1, by OriGene (1 mentions) Rpa70, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Bromodeoxyuridine (brdu), by BD (1 mentions) Tubulin, by Merck Group (1 mentions) Brca2, by Santa Cruz Biotechnology (1 mentions) Rpa32, by Abcam (1 mentions) Rad51, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions) Brca1, by Abcam (1 mentions) Dna ligase 4, by Abcam (1 mentions) Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ab81292, by Abcam (1 mentions) Phospho histone h2a x ser139, by Cell Signaling Technology (1 mentions)

6 protocols using mre11

Cell Lysis and Protein Immunoblotting Protocol

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Cells were harvested in PBS and lysed for 30 min on ice in IP lysis buffer (200 mM NaCl, 0.75% Chaps, 50 mM Tris pH 8.0) or RIPA buffer (10 mM Tris-Cl (pH 8.0) 1 mM EDTA. 1% Triton X-100. 0.1% sodium deoxycholate. 0.1% SDS. 140 mM NaCl) (Hall et al., 2014 (link)), supplemented with protease inhibitors. Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4°C) and the supernatants were collected. Protein samples were quantified with the Bradford assay and resolved by SDS-PAGE, transferred onto PVDF, and probed using the indicated primary antibodies. The membrane was stained with Alexa Fluor 680 or 800 anti-mouse/rabbit secondary antibody (Life Technologies) and visualized with the Licor Odyssey system. The following antibodies were used for staining: GAPDH (Santa Cruz, sc-47724); Flag (Santa Cruz, sc-51590); GFP (Abcam, Ab6556); HA (Sigma, H9658), RPA70 (Cell Signaling, 2267), RPA32 (Santa Cruz, sc-14692), SAMHD1 (Origene, TA502024), CtIP (14-1, a generous gift from Richard Baer) (Yu and Baer, 2000 (link)), BRCA2, RAD51 (Calbiochem, PC130), γH2AX (Cell Signaling, 2577 or Millipore, 05-636), 53BP1 (Bethyl, A300-273A), BrdU (BD Biosciences, 347580), MRE11 (Abcam, ab30725).

Daddacha W., Koyen A.E., Bastien A.J., Head P.E., Dhere V.R., Nabeta G.N., Connolly E.C., Werner E., Madden M.Z., Daly M.B., Minten E.V., Whelan D.R., Schlafstein A.J., Zhang H., Anand R., Doronio C., Withers A.E., Shepard C., Sundaram R.K., Deng X., Dynan W.S., Wang Y., Bindra R.S., Cejka P., Rothenberg E., Doetsch P.W., Kim B, & Yu D.S. (2017). SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination. Cell reports, 20(8), 1921-1935.

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Western Blot Analysis of DNA Repair Proteins

Cited 1 time

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Whole cell extracts and the protein concentration measurements for western blotting were performed as described previously (51 (link)). Equal amounts of protein were separated on NuPAGE Tris-Acetate or Bis-Tris gel (Invitrogen) and transferred to a PVDF membrane (0.45 μm pore size) (Invitrogen). After blocking, the membranes were incubated with appropriate primary antibodies at 4°C overnight followed by secondary antibodies at room temperature for 1 h. Imaging of protein was performed by the BIO-RAD ChemiDoc Imaging sytem.
Primary antibodies used for western blotting are listed below:
BRCA2 (Santa Cruz, sc-28235); BRCA1 (Calbiochem, OP92); RAD51 (Santa Cruz, sc-8349); CtIP (GeneTex 19E8); MRE11 (Abcam, ab33125); RPA32 (Abcam, 2175); Tubulin (Sigma, T5168). All secondary antibodies were purchased from Invitrogen.

Ahrabi S., Sarkar S., Pfister S.X., Pirovano G., Higgins G.S., Porter A.C, & Humphrey T.C. (2016). A role for human homologous recombination factors in suppressing microhomology-mediated end joining. Nucleic Acids Research, 44(12), 5743-5757.

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Chromatin-bound Protein Analysis

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Protein extracts (30 μg) were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and detected by incubations in primary antibodies followed by HRP-linked secondary antibodies at manufacturer's specifications. Chromatin-bound proteins were extracted from formaldehyde cross-linked (1%) cells using the Pierce Chromatin Prep module (Thermo Pierce, Rockford, IL, USA). Antibody sources: UNG-23936 (39 kDa band, nuclear UNG), Nbs1, DNA ligase IV, Ku70, BRCA1, MRE11, and PCNA (Abcam, Cambridge, MA, USA); Tubulin (Calbiochem, via Millipore, Billerica, MA, USA); γ-H2AX, cleaved caspase 3, and Histone-H3 (Millipore); cleaved PARP, XRCC4, and secondary antibodies (Cell Signaling, Danvers, MA, USA); and Rad51, p-chk1, chk1, p-cdc2, cdc2 (Santa Cruz, Dallas, TX, USA).

Weeks L.D., Zentner G.E., Scacheri P.C, & Gerson S.L. (2014). Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed. Cell Death & Disease, 5(2), e1045-.

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Protein Extraction and Analysis Protocol

Cited 2 times

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mRNA was extracted using TRIzol reagent (Invitrogen Corporation) and quantitative reverse transcription-PCR was performed as described.^{54 (link)} Primer sequences are given as Supplementary materials. Total protein extraction and western blot protocol have been previously described.^{54 (link)} Chromatin fractionation was performed as described.^{57 (link)} Primary antibodies were as follows: MYCN (B8.4.B), SC53993, p53 (DO-1) SC-126, CHK1 (G4), SC8408, Tubulin, (TU-02), SC-8035, and β-Actin (I-19) SC-1616 Santa Cruz Biotechnology; phospho-CHK1 (S317), DR1025, Calbiochem (San Diego, CA, USA); RAD50 (13B3/2C6), ab89, Mre11 (12D7) ab214, ZIC1 ab72694, Histone H3, ab1791, phospho-ATM (Ser 1981), ab81292, Rad50, ab499, and Mre11, ab6511, Abcam (Cambridge, UK); NBS1 (1C3) GTX70222, Gene Tex (Irvine, CA, USA); p53 (1C12), #2524, phospho-p53 (Ser 15), #9284, and phospho- histone H2AX (ser 139), #2577, Cell Signaling Technology (Danvers, MA, USA); PARP p85 fragment, #G7341, Promega Corporation; RPA32, A300-244 A, phospho-RPA32 S4/S8, A300-245 A, Bethyl Laboratories (Montgomery, TX, USA); Nbs1 (Y112), NB110-57272, Novus Biologicals; Immunoreactive bands were visualized by enhanced chemoluminescence (Advansta Inc., Menlo Park, CA, USA).

Petroni M., Sardina F., Heil C., Sahún-Roncero M., Colicchia V., Veschi V., Albini S., Fruci D., Ricci B., Soriani A., Di Marcotullio L., Screpanti I., Gulino A, & Giannini G. (2015). The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress. Cell Death and Differentiation, 23(2), 197-206.

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Nucleoprotein Extraction and Western Blotting

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Nucleoprotein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Total protein was extracted using radioimmunoprecipitation buffer supplemented with the protease inhibitor PMSF. Western blotting was performed as described previously (27 (link)). Each blot was repeated in three separate experiments. Band intensities were measured using Fusion software and the data are presented as relative protein levels normalized to GAPDH or histone H3. The antibodies used in this study were as follows: FoxM1 and P-RAD50 (Cell Signaling Technology, Danvers, MA, USA); Bax, Bcl2, GAPDH, NBS1, P-NBS1, MRE11, P-MRE11, RAD50, ATM, P-ATM, and γH2AX (Abcam, Cambridge, UK); and caspase-3, cleaved caspase-3, and histone H3 (Wanleibio, China).

Li D., Ye L., Lei Y., Wan J, & Chen H. (2019). Downregulation of FoxM1 sensitizes nasopharyngeal carcinoma cells to cisplatin via inhibition of MRN-ATM-mediated DNA repair. BMB Reports, 52(3), 208-213.

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Fluzoparib and Chidamide Inhibitor Protocol

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Fluzoparib is a PARP inhibitor and chidamide is a HDAC inhibitor. PARP, RAD51, MRE11, cleaved Caspase9, GAPDH, and P-CDK1 antibodies were obtained from Abcam Trading Co., Ltd. (Shanghai, China).

Li X., Yuan X., Wang Z., Li J., Liu Z., Wang Y., Wei L., Li Y, & Wang X. (2022). Chidamide Reverses Fluzoparib Resistance in Triple-Negative Breast Cancer Cells. Frontiers in Oncology, 12, 819714.

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