Phospho atm ser1981

Manufactured by Cell Signaling Technology

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Phospho-ATM Ser1981 is a laboratory reagent that specifically detects the phosphorylation of the ATM (Ataxia Telangiectasia Mutated) protein at serine 1981. This post-translational modification is a marker of ATM activation in response to DNA double-strand breaks.

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Phospho-ATM (18 citations) Anti-phospho-ATM (Ser1981) (6 citations) Rabbit anti-phospho-ATM (Ser1981) (2 citations)

21 protocols using phospho atm ser1981

Protein Expression Analysis Protocol

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Abs against phospho-ATM (Ser1981), ATM, phospho-Chk1 (Ser317), Chk1, phospho-Chk2 (Thr68), Chk2, phospho-p53 (Ser15), PTEN, phospho-CDK2 (Thr160), CDK2, Cyclin E, Cyclin B1, CDC2, phospho-CDC2 (Tyr15), cleaved PARP (Asp214), phospho-STAT3 (Ser727), STAT3, STAT5, caspase-3 and active caspase-3 (Asp175) were all obtained from Cell Signaling (Danvers, MA, USA). Abs recognizing p53 (EMD Millipore, Darmstadt, Germany), p21 (Santa Cruz, Dallas, Texas) and phospho-STAT5 (Ser726/731) (Sabbiotech, College Park, MD, USA) were obtained as indicated.

Chen L.M., Liu P.Y., Chen Y.A., Tseng H.Y., Shen P.C., Hwang P.A, & Hsu H.L. (2017). Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression. Scientific Reports, 7, 11864.

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Molecular Mechanisms of CPT-Induced Apoptosis

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The synchronized PC3 cells were incubated with 0.5 µM of CPT (Sigma), 0.5 µM of CFS-1686, and DMSO, respectively, for 0, 1, 2, 3, 4, and 5 hr. Then the cells were subjected to sonification and centrifuged to remove cell debris, and the supernatants were collected. Protein concentration was determined by a protein assay kit (PIERCE). About 40 µg of protein/per well was resolved in electrophoresis aparatus. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The transferred membranes were blocked in 5% (w/v) nonfat dry milk in TBST (0.5 M NaCl, 20 mM Tris-HCl, 0.05% (v/v) Tween 20, pH 7.4) and probed for antibody against ATM (Cell Signaling), Phospho-ATM (Ser-1981; Cell Signaling), γH2AX (Cell Signaling), PARP, caspase 3 activation form and GAPDH (Cell Signaling), followed by incubation with a secondary antibody conjugated horseradish peroxidase (anti-rabbit, anti-mouse, anti-goat; Jackson ImmunoResearch) and visualization with chemoluminiscence substrate (Millipore) using the chemoluminiscence detection system (BIO-RAD) or exposing to X-ray film.

Lin R.W., Yang C.N., Ku S., Ho C.J., Huang S.B., Yang M.C., Chang H.W., Lin C.M., Hwang J., Chen Y.L., Tzeng C.C, & Wang C. (2014). CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA. PLoS ONE, 9(12), e113832.

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Breast Cancer Cell Lines Protocol

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MCF7 and MDA-MB-231 breast cancer cell lines (ATCC; STR authenticated) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 100 Units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (PSG, Sigma-Aldrich) and 10% heat inactivated fetal bovine serum (FBS, First Link). MCF7/LMTK3 and MDA-MB-231/LMTK3, stably over-expressing LMTK3, were cultured in DMEM supplemented with 10% FCS, G418 (500 μg/ml; Invitrogen) and 1% PSG. All cells were incubated at 37 °C in humidified 5% CO₂, and were frequently tested for mycoplasma contamination. Antibodies used: LMTK3 (sc-100418, Santa Cruz Biotechnology); LMTK3 (H00114783-M02, Abnova); β-actin (ab627, Abcam); phospho-histone H2A.X Ser139 (2577, Cell Signaling Technology); phospho-histone H2A.X Ser139 (9718, Cell Signaling Technology); ATM (sc-23921, Santa Cruz Biotechnology); phospho-ATM Ser1981 (4526, Cell Signaling Technology); KAP1 (ab10484, Abcam); phospho-KAP1 Ser824 (ab70369, Abcam). Doxorubicin hydrochloride (D1515) was purchased from Sigma-Aldrich.

Stebbing J., Shah K., Lit L.C., Gagliano T., Ditsiou A., Wang T., Wendler F., Simon T., Szabó K.S., O’Hanlon T., Dean M., Roslani A.C., Cheah S.H., Lee S.C, & Giamas G. (2018). LMTK3 confers chemo-resistance in breast cancer. Oncogene, 37(23), 3113-3130.

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Western Blot Analysis of Cell Signaling

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Western blot analysis was performed using standard procedures for whole-cell extracts from cell lines. Antibodies used include Chk2 (1:5000, Becton Dickinson and Millipore), SIRT1 (1:2000, Millipore), acetyl-lysine (1:1000), phospho-CHK2-T68 (1:1000), phospho-CHK2-Thr387 (1:1000), phospho-p53-Ser20 (1:1000), acetyl-p53-Thr382 (1:1000), phospho-histone H2A.X (Ser139) (1:1000), phospho-ATM-Ser1981 (1:1000), ATM (1:1000), phospho-histone H3-S10 (1:1000), p-CDC25C (ser216) (1:1000), CDC25C (1:1000), cleaved PARP-1 (1:1000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology), phospho-CHK2-Thr432 (1:500, Invitrogen), P53 (Do-1, 1:1000, Santa Cruz Biotechnology), FLAG (clone M2) (1:2000), α-tubulin (1:5000, Sigma), and β-actin (1:5000, Sigma). For immunoprecipitation analysis, cell lysates (1–4 mg) after preclearing were mixed with antibodies (2 μg) at 4 °C overnight followed by the addition of 30 μl of protein-G (for mouse antibodies)- or protein-A (for rabbit antibodies)-coupled sepharose beads (GE) for 3 h at 4 °C. Immune complexes were washed three times with lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, protease, phosphatase inhibitor mixture (Sigma)]. After boiling in 2× loading buffer, samples were subjected to SDS-PAGE, and then scanned using ECL.

Zhang W., Feng Y., Guo Q., Guo W., Xu H., Li X., Yi F., Guan Y., Geng N., Wang P., Cao L., O’Rourke B.P., Jo J., Kwon J., Wang R., Song X., Lee I.H, & Cao L. (2019). SIRT1 modulates cell cycle progression by regulating CHK2 acetylation−phosphorylation. Cell Death and Differentiation, 27(2), 482-496.

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Comprehensive Western Blot Analysis

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Western blotting was performed as previously described according to the standard protocol [57 ,58 (link)]. The primary antibodies and concentrations used for western blotting were: PARP1 (Cell Signaling Technology, 1:1000 dilution), SLC7A11/xCT (D2M7A) (Cell Signaling Technology, 1:1000 dilution), p53 (Cell Signaling Technology, 1:1000), GPX4 (Abcam-ab125066, 1:1000 dilution), BAP1 (Cell Signaling Technology, 1:1000), ATF3 (Cell Signaling Technology, 1:1000), NRF2 (Cell Signaling Technology, 1:1000), Vinculin (Cell Signaling Technology, 1:3000), Tubulin (Cell Signaling Technology, 1:3000), phospho-histone H2A.X (Ser139) (Cell Signaling Technology, 1:1000), phospho-Chk2 (Thr68) (Cell Signaling Technology, 1:1000), phospho-ATM (Ser1981) (Cell Signaling Technology, 1:1000), cleaved Caspase-3(Cell Signaling Technology, 1:1000). The secondary antibodies used were: horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, 1:5000 dilution), horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology, 1:5000 dilution). Proteins were visualized with the ECL Western blotting substrate (32,109, ThermoScientific, USA).

Hong T., Lei G., Chen X., Li H., Zhang X., Wu N., Zhao Y., Zhang Y, & Wang J. (2021). PARP inhibition promotes ferroptosis via repressing SLC7A11 and synergizes with ferroptosis inducers in BRCA-proficient ovarian cancer. Redox Biology, 42, 101928.

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Cucurbitacin B Molecular Signaling

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Cucurbitacin B, purchased from ShunBo Biological Engineering Technology Co., Ltd (Shanghai China), was dissolved in dimethyl sulfoxide (DMSO) to make 200 µM stock solutions and was kept at −20°C. The stock solutions were freshly diluted to the desired concentration just before use. N-acetyl-L-cysteine (NAC) was purchased from Beyotime (Haimen, China). Specific antibodies against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phospho-p53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA). Phospho-Cdk1-Tyr-15, Cyclin B1, 14-3-3-σ were from Santa Cruz Biotechnology (USA). Cdk1 antibody was obtained from BD Transduction Laboratories™ (USA). Antibodies for ATM and γH2AX were obtained from GeneTex and Millipore respectively.

Guo J., Wu G., Bao J., Hao W., Lu J, & Chen X. (2014). Cucurbitacin B Induced ATM-Mediated DNA Damage Causes G2/M Cell Cycle Arrest in a ROS-Dependent Manner. PLoS ONE, 9(2), e88140.

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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 Antibodies for DNA Damage Response

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The following antibodies were used for immunoblotting: rabbit polyclonal antisera raised against SMC1 (BL308; Bethyl Laboratories), BRCA1 (Bouwman et al, 2010 (link)), 53BP1 (NB100-304; Novus Biologicals), PARP1 (46D11; Cell Signaling) and human histone H3 (a gift from Dr. Alain Verreault, University of Montreal); mouse monoclonal antibodies raised against TRF2 (NB100-57130; Novus Biologicals), phospho-ATM Ser1981 (Cell Signaling), MRE11 (NB100-142; Novus Biologicals), CHK2/Cds1 (clone 7, Upstate), CtIP (a gift from Dr. Richard Baer, Columbia University), NBS1 (ab49958; Abcam), WRN (a gift from Dr. Thomas Helleday, Karolinska Institute), GAPDH (NB600-502; Novus Biologicals), LIG3 (611876; BD Transduction) and α-tubulin (Cancer Research UK Monoclonal Antibody Service). In addition, rabbit polyclonal antibodies raised against RAD51 (FBE2; Clare Hall Laboratories) and 53BP1 (NB100-304; Novus Biologicals) as well as mouse monoclonal against phosphorylated histone H2AX-Ser139 (JBW301; Upstate) were used for immunofluorescence detection.

Badie S., Carlos A.R., Folio C., Okamoto K., Bouwman P., Jonkers J, & Tarsounas M. (2015). BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres. The EMBO Journal, 34(3), 410-424.

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Western Blot Protein Quantification

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Western blot analysis was carried out as previously described [30 ]. Primary antibodies used in western blot were listed as below: PLK4 (#NBP1-33042, Novus, USA), LATS1 (#3477, Cell Signaling Technology, USA), phospho-LATS1 (Thr1079) (#8654, Cell Signaling Technology), YAP (#14074, Cell Signaling Technology), phospho-YAP (Ser127) (#13308, Cell Signaling Technology), cleaved PARP (#5625, Cell Signaling Technology), γ-H2AX (Ser139) (#9718, Cell Signaling Technology), phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), phospho-ATR (Ser428) (#2853, Cell Signaling Technology), phospho-Chk1(Ser345) (#2348, Cell Signaling Technology), phospho-Chk2 (Thr68) (#2197, Cell Signaling Technology), anti-Histone H3 (#4499, Cell Signaling Technology). β-actin was used as a loading control. Chemiluminescent signals were detected using the Amersham Imager 600 imaging system (General Electric, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein bands normalized to control.

Zhao Y., Yang J., Liu J., Cai Y., Han Y., Hu S., Ren S., Zhou X, & Wang X. (2021). Inhibition of Polo-like kinase 4 induces mitotic defects and DNA damage in diffuse large B-cell lymphoma. Cell Death & Disease, 12(7), 640.

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Immunoblotting Analysis of Cellular Proteins

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The whole cell lysate were prepared for immunoblotting as described previously [27 (link)]. Antibodies for P53, phospho-P53 (Ser15), P21CIP1, cleaved-PARP (c-PARP), cleaved caspase-3, SRC-1, SRC-3, HK2, phospho-ATM (Ser1981), and phospho-H2A.X (Ser139) were obtained from Cell Signaling Technology (Danvers, MA). Antibody for LC3 was purchased from MBL (Woburn, MA). Antibody for AR was obtained from Millipore (Billerica, MA) and PSA antibody was obtained from DAKO (Glostrup, Denmark). Antibody for β-actin was obtained from Sigma-Aldrich (St. Louis, MO). Positive and negative controls were included in immunoblot analyses whenever possible.

Zhang Y., Dong Y., Melkus M.W., Yin S., Tang S.N., Jiang P., Pramanik K., Wu W., Kim S., Ye M., Hu H., Lu J, & Jiang C. (2018). Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin. Molecular cancer therapeutics, 17(11), 2341-2352.

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