Phospho chk2 thr68

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-Chk2 (Thr68) is a lab equipment product that detects the phosphorylation of the Chk2 protein at the Thr68 residue. This phosphorylation event is a key step in the activation of the Chk2 protein, which plays a crucial role in the cellular response to DNA damage.

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Close spelling variants of this product

Phospho-Chk2 (29 citations) Anti-phospho-Chk2 (Thr68) (19 citations) Rabbit anti-phospho-Chk2 (Thr68) (4 citations) Chk2 (Thr68) (2 citations) Phospho-CHK2(Thr68) (2661) (2 citations)

36 protocols using phospho chk2 thr68

Western Blot Analysis of DNA Damage Response

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The details are described in previous report [32 (link)]. For Western blotting, the used antibodies were as following: p21 (OP79; Oncogene Science), p-ATM (Ser1981) (sc-47739; Santa Cruz, CA, USA), p53 (sc-98, Santa Cruz), cleaved caspase-3 (#9661; Cell Signaling, Danvers, MA, USA), phospho-Chk2 (Thr68) (#2197; Cell Signaling), Chk2 (#05-649; EMD Millipore, Temecula, CA, USA). LC3A/B (#12741; Cell Signaling), Chk1 (#2345; Cell Signaling), phospho-Chk1 (Ser345) (#2348; Cell Signaling), phospho-Histone H2A.X (Ser139) (#9718; Cell Signaling), ATM (GTX70103; GeneTex, Irvine, CA, USA), GAPDH (#2118, Cell Signaling), PARP (#9542; Cell Signaling), phospho-p53 (Ser15) (#9284; Cell Signaling). phospho-ATR (Ser428) (#2853; Cell Signaling). PAI-1 (#612024; BD Transduction Laboratories, Lexington, KY, USA).

Yang S.C., Chen H.Y., Chuang W.L., Wang H.C., Hsieh C.P, & Huang Y.F. (2022). Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines. International Journal of Molecular Sciences, 23(3), 1632.

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Western Blot Analysis of DNA Damage Response

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Western blotting was performed as previously described [49 (link)]. Briefly, equal amounts of lysates were resolved with SDS-PAGE and were transferred onto PVDF membranes. Membranes were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal amplification and detection were achieved by exposing the membrane to enhanced chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK), followed by visualization using the Storm imaging system (Amersham Biosciences, Piscataway, NJ, USA). The following primary antibodies were used: γ-H2AX (1/1000; Cell Signaling Technology, Beverly, MA, USA), Chk2 (1/500; Cell Signaling Technology), Phospho-Chk2 (Thr68) (1/1000; Cell Signaling Technology), XRCC2 (1:1500; Abcam), Caspase-9 (1/500; Proteintech, Chicago, IL, USA), Caspase-3 (1/500; Proteintech), PARP (1/500; Proteintech), and BCL-2 (1/500; Santa Cruz Biotechnology). Detection of GADPH (1/10000; Cell Signaling Technology) was used as a loading control. Bound antibodies were visualized with peroxidase-linked secondary antibodies (anti-rabbit antibody: 1/10000; Cell Signaling Technology and anti-mouse antibody: 1/5000; Sigma-Aldrich, St. Louis, MO, USA).

Qin C.J., Song X.M., Chen Z.H., Ren X.Q., Xu K.W., Jing H, & He Y.L. (2015). XRCC2 as a predictive biomarker for radioresistance in locally advanced rectal cancer patients undergoing preoperative radiotherapy. Oncotarget, 6(31), 32193-32204.

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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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Analysis of DNA Damage Response

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Cells were lysed using cell lysis buffer (Cell Signaling) with complete protease inhibitor cocktail (1:1,000, Roche) and Halt phosphatase inhibitor cocktail (1:100, Thermo). After total protein quantification using a Bradford reagent (Bio-Rad), 20 μg of cell lysate protein was resolved by 4–15% SDS-PAGE (Bio-Rad) under denaturing conditions, and transferred to PVDF membranes. Membranes were blocked in 5% BSA and then incubated with anti- phospho-H2AX (1:1,000, Cell Signaling), phospho-Chk1-Ser³¹⁷ (1:1,000; Cell Signaling), phospho-Chk2-Thr⁶⁸ (1:1,000, Cell Signaling), caspase-3 (1:1,000; Cell Signaling), cleaved caspse-3 (1:1,000; Cell Signaling), PARP (1:1,000; Cell Signaling), and GAPDH (1:5,000, Cell Signaling) antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (HRP) (1:2,000; Cell Signaling). Signals were visualized using ECL Prime Western Blotting Detection reagent (Amersham).

Alan Mitteer R., Wang Y., Shah J., Gordon S., Fager M., Butter P.P., Jun Kim H., Guardiola-Salmeron C., Carabe-Fernandez A, & Fan Y. (2015). Proton beam radiation induces DNA damage and cell apoptosis in glioma stem cells through reactive oxygen species. Scientific Reports, 5, 13961.

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Antibody Panel for DNA Damage Response

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The antibodies used were phospho-CHK2-Thr-68 (Cell Signaling; catalog no. 2661), SMC1 (Abcam, Inc.; catalog no. ab9262), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), RAD51 (Millipore; catalog no. 05-530-I), BRCA1 (Thermo Fisher; catalog no. MA1-23164), γ-H2AX (Millipore; catalog no. 05-636), normal mouse IgG (Santa Cruz; catalog no. SC-2025), bromodeoxyuridine (BrdU) (Abcam, Inc.; catalog no. Ab-8039), and anti-DNA-RNA hybrid antibody, clone S9.6 (Millipore; catalog no. MABE1095).

Mehta K, & Laimins L. (2018). Human Papillomaviruses Preferentially Recruit DNA Repair Factors to Viral Genomes for Rapid Repair and Amplification. mBio, 9(1), e00064-18.

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Molecular Signaling Profiling by Western Blot

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Western blot was performed following a standard protocol. Antibodies were obtained as follow; p53, p21, Chk1, p62, PARP-1, β-actin (Santa Cruz Biotechnology), phospho-p53 (Ser15), phospho-Chk2 (Thr68), phospho-Erk1/2, Erk1/2, phospho-JNK, JNK1/2, phospho-p38, p38, Caspase 3, H2AX (Cell Signaling Technology), phospho-ATM (Ser1981), Chk2, γ-H2AX (Ser139) (Millipore), ATM (Epitomics, Burlingame, CA, USA), and LC3 (Novus Biologicals, Littleton, CO, USA). Chemiluminescence was detected using enhanced chemiluminescence detection reagents.

Kang M.A., Kim M.S., Kim W., Um J.H., Shin Y.J., Song J.Y, & Jeong J.H. (2016). Lanatoside C suppressed colorectal cancer cell growth by inducing mitochondrial dysfunction and increased radiation sensitivity by impairing DNA damage repair. Oncotarget, 7(5), 6074-6087.

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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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Western Blot Analysis of DNA Damage Signaling

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Cell pellets were lysed in RIPA lysis buffer and the protein content was determined using a DC protein assay kit (Bio-Rad Laboratories). The proteins were run on electrophoresis gels and transferred to nitrocellulose membranes (GE Osmonics Labstore) as described previously. [24 (link)] Membranes were blocked for 1 hour in blocking buffer, incubated with primary antibodies against total ATM (ab17995; Abcam, Cambridge, MA), phospho-ATM^Ser1981 (EMD Millipore, Billerica, MA), total p53 (EMD Millipore, Billerica, MA), phospho-p53^Ser15 (05-740; Cell Signaling Technology, Danvers, MA), CHK-2 (3440) and phospho-CHK-2^Thr68 (2661; Cell Signaling Technology, Danvers, MA), MCL-1 (AHO0102; Life Technologies Corporation, Carlsbad, CA), PUMA (3043; ProSci Incorporated, Poway CA), BAX (sc-20067; Santa Cruz Inc, Dallas, TX) and GAPDH (5174; Cell Signaling Technology, Danvers, MA).
Following washing with PBST, membranes were incubated for 1 hour with infrared dye-labeled secondary antibodies (LI-COR Biosciences), scanned, and visualized using a LI-COR Odyssey infrared imager.

Jain N., Balakrishnan K., Ferrajoli A., O’Brien S.M., Burger J.A., Kadia T.M., Cortes J.E., Ayres M.L., Tambaro F.P., Keating M.J., Gandhi V, & Wierda W.G. (2016). A phase I-II trial of fludarabine, bendamustine and rituximab (FBR) in previously treated patients with CLL. Oncotarget, 8(13), 22104-22112.

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Western Blot Analysis of Cellular Proteins

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Western blot analysis was performed on total cell extracts using the NuPAGE system (Thermo Fisher Scientific) or the Mini PROTEAN TGX gels and the Trans-Blot Turbo Transfer System (Biorad). The membrane was blocked with 4% milk solution and blotted with primary antibodies. Antibodies used were: vimentin (Sigma-Aldrich Cat# V6630, RRID:AB_477627; diluted 1:400), GAPDH (Sigma-Aldrich Cat# SAB1405848, RRID:AB_10739020; diluted 1:1000), p53-DO7 (Santa Cruz Biotechnology Cat# sc-47698, RRID:AB_628083; 1:500), cleaved PARP (Cell Signaling Technology Cat# 5625, RRID:AB_10699459; 1:1000), phospho-Histone H2A.X (Ser139) (Millipore Cat# 05-636, RRID:AB_309864; 1:1000), H2AX (Millipore Cat# DR1016-100UG, RRID:AB_437862; 1:1000), vinculin (Sigma-Aldrich Cat# V9264, RRID:AB_10603627; 1:1000), Phospho-Chk1 (ser317) (Cell Signaling Technology Cat# 12302, RRID:AB_2783865; 1:1000), CHK1 (Cell Signaling Technology Cat# 2360, RRID:AB_2080320; 1:1000), β-Actin (Sigma-Aldrich Cat# A1978, RRID:AB_476692; 1:4000), Phospho-Chk2 (Thr68) (Cell Signaling Technology Cat# 2661 (also 2661S, 2661P, 2661L, 2661T), RRID:AB_331479; 1:1000), CHK2 (Abcam Cat# ab109413, RRID:AB_10863751; 1:50000). The results were quantified by densitometry using Fiji software. Experiments were repeated at least two times.

Celli L., Gasparini P., Biino G., Zannini L, & Cardano M. (2024). CRISPR/Cas9 mediated Y-chromosome elimination affects human cells transcriptome. Cell & Bioscience, 14, 15.

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Western Blot Analysis of DNA Repair Proteins

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Cells were harvested by trypsinization, lysed in 2X Laemmli buffer (100 mM Tris-HCl pH 6.8, 200 µM DTT, 3% SDS, 20% glycerol, 0.05% bromophenol blue) at 10⁴ cell/µl. The lysate was denatured for 10 min at 95°C, and sheared by forcing it through a 28-gauge insulin needle 10 times. Lysate from 10⁵ cells was loaded on an SDS/PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in 5% milk in TBS with 0.1% Tween-20 and incubated with primary antibody in TBS/5% milk/0.1% Tween-20 for 2 hours at room temperature. The following primary antibodies were utilized: Polθ (ab80906, Abcam); TRF1 (1449, rabbit polyclonal); Rap1 (1252, rabbit polyclonal); Phospho-Chk2 (Thr68) (Rabbit polyclonal, Cell Signaling); Chk2 (rabbit polyclonal, Cell Signaling); Phospho-Chk1 (Ser 345) (mouse monoclonal, Cell Signaling); Chk1 (mouse monoclonal, Santa Cruz); Lig3 (mouse monoclonal, Santa Cruz); Myc (9E10; Calbiochem); γ- tubulin (clone GTU-88, Sigma); PARP1 (Polyclonal, Cell signaling). (From http://delangelab.rockefeller.edu/protocols)

Mateos-Gomez P.A., Gong F., Nair N., Miller K.M., Lazzerini-Denchi E, & Sfeir A. (2015). Mammalian Polymerase Theta Promotes Alternative-NHEJ and Suppresses Recombination. Nature, 518(7538), 254-257.

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