P atm

Manufactured by Cell Signaling Technology

Sourced in United States

The P-ATM is a laboratory equipment product that detects and measures the phosphorylation of the ATM protein. It is used for research purposes in the field of cell signaling and DNA damage response pathways.

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P-ATM (Ser1981) (22 citations) Anti-p-ATM (15 citations) P-ATM S1981 (15 citations) P-Aurora A/B/C (8 citations) P∼Aurora-A (3 citations) Anti-p-Aurora A (Thr288) (2 citations)

50 protocols using p atm

Western Blot Analysis of DNA Damage Signaling

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Cells were seeded in 6-well plates and allowed to attach for 24 h. Cells were then incubated for 24 hours with indicated concentrations of AZ31 and/or SN38. Cells were then washed with PBS and lysed with RIPA buffer (Cell Signaling, Danvers, MA). After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel (Life Technologies, Carlsbad, CA), electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter (Thermo Fisher, Rockford, IL). The membrane was blocked and probed overnight with primary antibodies (Cell Signaling Technologies (Danvers, MA) at a concentration 1:1000: p-ATM (catalog# 13050), ATM (catalog# 2873), p-p53 (catalog# 9284), p53 (catalog# 2527), p-CHK2 (catalog# 2665), p-RAD50 (catalog# 14223), p-H2AX (catalog# 9718) and actin (catalog# 4970). The next day the membranes were washed for 10 minutes 3X with TBS/Tween 20, and then probed with DyLight secondary antibodies 1:15,000 (Cell signaling, Danvers, MA), and imaged using the Licor Odyssey (Licor, Lincoln, NE).

Greene J., Nguyen A., Bagby S.M., Jones G.N., Tai W., Quackenbush K.S., Schreiber A., Messersmith W.A., Devaraj K.M., Blatchford P., Eckhardt S.G., Cadogan E.B., Hughes G.D., Smith A., Pitts T.M, & Arcaroli J.J. (2017). The novel ATM inhibitor (AZ31) enhances antitumor activity in patient derived xenografts that are resistant to irinotecan monotherapy. Oncotarget, 8(67), 110904-110913.

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Resveratrol-Induced DNA Damage Response

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Cells were cultured and treated with resveratrol for different time periods and different concentrations. Next, cells were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mg/mL PMSF, 2 mg/mL aprotinin, 2 mM leupeptin, and 1 mg/mL pepstatin) and protein concentration was determined using the Bradford assay. Protein extracts were resolved by SDS–PAGE (60 mg per lane) on a 10% polyacrylamide gel and transferred into immobilon membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, membranes were incubated with 1:1000 dilutions of primary antibodies. We used antibodies against p-ATM, p-BRCA1 and γ-H2AX (Cell Signaling Technology, Inc., Danvers, MA, USA) as markers of DNA damage response; active-caspase 3 and cleaved PARP (Cell Signaling Technology, Inc., Danvers, MA, USA) to determine apoptosis marker; and finally, Rad50, Mre11, p-p95/NBS1, DNA-PKcs, and KU80 (Cell Signaling Technology, Inc, Danvers, MA, USA) as markers of DNA repair; Tubulin (Calbiochem, Darmstadt, Germany) was used as loading control.

Jara P., Spies J., Cárcamo C., Arancibia Y., Vargas G., Martin C., Salas M., Otth C, & Zambrano A. (2017). The Effect of Resveratrol on Cell Viability in the Burkitt’s Lymphoma Cell Line Ramos. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(1), 0014.

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Western Blot Analysis of DNA Damage Proteins

Cited 2 times

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CD4 T cells (2 × 10⁶) purified from HCV, HBV, or HIV patients and HS were used for western blot as described previously [26 (link), 27 (link)]. Primary and secondary antibodies included Top1, PARP1, γH2AX, TRF1, TRF2, TPP1, TIN2, RAP1, POT1, ATM, pATM, CHK2, ATR, GAPDH, β-actin, and horseradish peroxide-conjugated antibodies (Cell Signaling). Images were captured using ChemiDoc™ XRS+ System (Bio-Rad). Protein band intensity was quantitated by the Image Lab software (Bio-Rad).

Ji Y., Dang X., Nguyen L.N., Nguyen L.N., Zhao J., Cao D., Khanal S., Schank M., Wu X.Y., Morrison Z.D., Zou Y., El Gazzar M., Ning S., Wang L., Moorman J.P, & Yao Z.Q. (2019). Topological DNA damage, telomere attrition and T cell senescence during chronic viral infections. Immunity & Ageing : I & A, 16, 12.

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Evaluation of DNA Damage Signaling

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CDDP, 5-fluorouracil (5-Fu) and MN were purchased from Selleck Chemicals (Houston, USA). Antibodies specific for the following proteins were used in this study were as follow: p-ATM, p-Chk1, p-Chk2, cleaved-PARP, cleaved-caspase 3, Vinculin (all from Cell Signaling Technology, Beverly, MA, USA), Lamin B1, FH, ATM, Chk1, Chk2, γ-H2AX (all from Abcam, Cambridge, UK), GAPDH (Sigma-Aldrich, St Louis, USA), DNA-PK (Affinity Biosciences, Cincinnati, OH, USA) and Ki-67 (Zhongshan Golden Bridge Biotech, Beijing, China).

Yu H.E., Wang F., Yu F., Zeng Z.L., Wang Y., Lu Y.X., Jin Y., Wang D.S., Qiu M.Z., Pu H.Y., Kang T.B., Xie D., Ju H.Q., Xu R.H, & Luo H.Y. (2019). RETRACTED ARTICLE: Suppression of fumarate hydratase activity increases the efficacy of cisplatin-mediated chemotherapy in gastric cancer. Cell Death & Disease, 10(6), 413.

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Western Blotting Analysis of DNA Damage Signaling

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After being washed with ice-cold PBS, cells were lysed with RIPA Lysis Buffer (VWRVN653-100ML, VWR Life Science), supplemented with Phosphatase Inhibitor Cocktail 2 (P5726-1ML, Sigma) and Protease Inhibitor Cocktail (B14001, Bimake). The Pierce BCA Protein Assay Kit (89167–794, Thermo Scientific) was used to determine protein concentration. Equal protein lysates were run on Novex 3–8% Tris-acetate 15 Well Mini Gels (EA03785BOX, Invitrogen) and transferred to Immobilon-P membranes (IPVH00010, Fisher Scientific). Membranes were then probed with the following primary antibodies: GAPDH (sc-47724, Santa Cruz Biotechnologies, 1:1000) and phospho DNA-PKcs S2056 (ab18192, Abcam). P300 (sc-48343, Santa Cruz Biotechnologies), pATM (13050S, Cell signaling), ATM (92356S, Cell Signaling), pATR (58014S, Cell signaling), and ATR (2790S, Cell signaling). After exposure to the matching HRP-conjugated secondary antibody, cells were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Scientific).

Hu C., Bugbee T., Dacus D., Palinski R, & Wallace N. (2022). Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors. PLoS Pathogens, 18(2), e1010275.

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Evaluation of Apoptosis and DNA Damage

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CD34⁺CD38⁻ KG1α cells (5 × 10⁵/ml) were cultured for 24 or 48 h in the absence or presence of 40 nM IDA and 0.75 μM chidamide. The protein expression levels were determined by staining with primary antibodies and relevant HRP-conjugated secondary (1:10,000, Abcam, Cambridge, UK) antibodies. The primary antibodies (caspase-3, caspase-8, caspase-9, and PARP—Beyotime, China; p-BRCA1, p-ATM, p-CHK1, p-CHK2, γH2A.X, and Ace-H3—Cell Signaling, Herts, UK) were diluted at 1:1000 in 5% fat-free milk-TBST. Anti-β-actin (1:1000, Cell Signaling, Herts, UK) was used as a loading control. The signal was detected using an ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK).

Li Y., Wang Y., Zhou Y., Li J., Chen K., Zhang L., Deng M., Deng S., Li P, & Xu B. (2017). Cooperative effect of chidamide and chemotherapeutic drugs induce apoptosis by DNA damage accumulation and repair defects in acute myeloid leukemia stem and progenitor cells. Clinical Epigenetics, 9, 83.

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Synthesis and Characterization of Anticancer Agents

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MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).

Tu H.J., Lin Y.J., Chao M.W., Sung T.Y., Wu Y.W., Chen Y.Y., Lin M.H., Liou J.P., Pan S.L, & Yang C.R. (2018). The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells. Clinical Epigenetics, 10, 162.

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Evaluating DNA Damage Response via IF and Comet

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Immunofluorescence (IF) was performed based on manufacturer instructions. Cells were: washed in PBS; fixed for 20 minutes at RT in 4% PFA; blocked for 1 hour at room temperature in 5% goat serum and 1% Triton X-100 in 1× PBS; incubated with primary antibody overnight at 4 degrees in 1% goat serum and 1% Triton X-100 in 1× PBS antibody diluent; incubated with secondary antibody in diluent for 1 hour at RT; and then mounted with DAPI-containing mounting media (cat#P36935). Primary antibodies used include pHistoneH3 (Cell Signaling; 1:500), pATM (cat#ab36810), pChk2 (cat#21997), MLH1 (cat#WH0004292M2). Cells were treated with fulvestrant for 48 hours before evaluation. Alkaline comet assay was performed as per the manufacturer’s instructions on a CometAssay Electrophoresis SystemII (Trevigen, cat#4250-050-ES). Calculation of nuclei with DNA damage was performed using CASPLab software²⁷ to calculate the ratio of DNA content in tail/head. Cut-offs for categorization were set as 0–0.5, 0.5–1, 1–5 and >5 for No, Low, Med and High DNA damage. Fluorescent images were captured with a Nikon microscope and quantified with ImageJ.

Haricharan S., Punturi N., Singh P., Holloway K.R., Anurag M., Schmelz J., Schmidt C., Lei J.T., Suman V., Hunt K., Olson JA J.r., Hoog J., Li S., Huang S., Edwards D.P., Kavuri S.M., Bainbridge M.N., Ma C.X, & Ellis M.J. (2017). Loss of MutL disrupts Chk2-dependent cell cycle control through CDK4/6 to promote intrinsic endocrine therapy resistance in primary breast cancer. Cancer discovery, 7(10), 1168-1183.

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Quantification of Cellular Proteins

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Total protein was extracted from exponentially growing cells. A total of 40 µg of protein was used for SDS-PAGE with a 4–15% gradient gel (Bio-Rad Laboratories, Hercules, CA, USA). After fast transfer (Trans-Blot Turbo Transfer System, Bio-Rad Laboratories) and blocking in 5% bovine serum albumin (BSA) for at least 1 h, proteins were detected using the following primary antibodies: USP7 (Abcam, Cambridge, UK) #ab108931, 1:1000), HSC70 (Santa Cruz, Dallas, TX, USA #sc7298, 1:1000), ATM (Cell Signaling, Danvers, MA, USA #2873, 1:1000) and pATM (Cell Signaling #4526, 1:500). Primary antibodies were marked with IRDYE 680 conjugated anti-mouse IgG, IRDYE 800 conjugated anti-rabbit IgG (LI-COR, Lincoln, NE, USA, 1:7, 500).

Vogt M., Classen S., Krause A.K., Peter N.J., Petersen C., Rothkamm K., Borgmann K, & Meyer F. (2024). USP7 Deregulation Impairs S Phase Specific DNA Repair after Irradiation in Breast Cancer Cells. Biomedicines, 12(4), 762.

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Investigating DNA Damage Responses in HepG2 Cells

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Pine needle oil [dissolved in dimethyl sulfoxide (DMSO), ≤0.1%], RNase and propidium iodide (PI) solution were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-γ-H2AX (cat no. 05636; 1:1,000) antibody was purchased from EMD Millipore Billerica, MA, USA), ATM (cat no. 2873; 1:500), p-ATM (cat no. 13050; 1:1,000), p-p53 (S15; cat no. 9286; 1:1,000), p-CDC25C (S216; cat no. 4901; 1:500) p-CHK2 (T68; cat no. 2661; 1:1,000) and CHK2 (cat no. 2662; 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). CDC25C (cat no. sc-327; 1:1,000), β-actin (cat no. sc-47778; 1:1,000), anti-H2AX (cat no. sc-54606; 1:200), p53 (cat no. sc-98; 1:500) antibodies, goat anti-rabbit (cat no. sc-2030; 1:3,000) and anti-mouse secondary (cat no. sc-2031; 1:3,000) antibodies were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Protein extraction solution kit was purchased from Beijing SBS Genetech Co., Ltd., (Beijing, China). Dulbecco's modified Eagle's medium and bovine serum albumin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HepG2 cell line was purchased from the China Center for Type Culture Collection of Wuhan University (Wuhan, China). This cell line was originally thought to be a hepatocellular carcinoma, but is now known to be a hepatoblastoma cell line (21 (link)).

Qiu B., Jiang W., Qiu W., Mu W., Qin Y., Zhu Y., Zhang J., Wang Q., Liu D, & Qu Z. (2017). Pine needle oil induces G2/M arrest of HepG2 cells by activating the ATM pathway. Experimental and Therapeutic Medicine, 15(2), 1975-1981.

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