Anti chk1 ps345

Manufactured by Cell Signaling Technology

Anti-CHK1 pS345 is a primary antibody that detects checkpoint kinase 1 (CHK1) phosphorylated at serine 345. It is used in research applications to study the activation and regulation of the CHK1 signaling pathway.

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Lab products found in correlation

Anti chk1, by Santa Cruz Biotechnology (4 mentions) Anti rad17, by Santa Cruz Biotechnology (2 mentions) Anti tubulin, by Merck Group (2 mentions) Anti p53, by Cell Signaling Technology (2 mentions) Anti p53 ps15, by Cell Signaling Technology (2 mentions) Ab81292, by Abcam (2 mentions) Ab18192, by Abcam (2 mentions) Gemcitabine, by Merck Group (1 mentions) Doxorubicin, by Merck Group (1 mentions) Mouse anti pcna, by Santa Cruz Biotechnology (1 mentions) Anti fancd2, by Santa Cruz Biotechnology (1 mentions) Anti chk2, by Abcam (1 mentions) Anti chk1 ps317, by Cell Signaling Technology (1 mentions) Goat anti atr, by Santa Cruz Biotechnology (1 mentions) Anti mcm2, by Fortis Life Sciences (1 mentions) Rabbit anti fancd2, by Abcam (1 mentions) Anti rad9, by Abcam (1 mentions) Rabbit anti atm ps1981, by Cell Signaling Technology (1 mentions) Hydroxyurea hu, by Merck Group (1 mentions) Etoposide, by Merck Group (1 mentions)

7 protocols using anti chk1 ps345

Synthesis and Antibody Validation for DNA Damage Response

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Hairpin Py–Im polyamides 1 and 2 were synthesized on solid phase Kaiser oxime resin using previously published protocols (18 (link)). Gemcitabine, etoposide, hydroxyurea (HU) and doxorubicin were purchased from Sigma-Aldrich, as were all other reagents unless otherwise noted.
Antibodies purchased from Santa Cruz Biotech were: mouse anti-PCNA, anti-Chk1, anti-RPA2, anti-Rad17, anti-FANCD2, goat anti-ATR and rat anti-BrdU (CldU cross-reactivity). Antibodies purchased from Bethyl were: rabbit anti-H2AX, anti-MCM2, anti-MCM2pS108 and anti-RPA2pS4/S8. Antibodies purchased from Abcam were: rabbit anti-FANCD2, anti-MCM2pS108, anti-Rad9, anti-RPA2pS33, anti-Chk2 and anti-H2AXpS139. Antibodies purchased from Cell Signaling Technologies were: rabbit anti-ATMpS1981, anti-Rad17pS645, anti-Chk1pS345, anti-Chk1pS317 and anti-Chk1pS296. Rabbit anti-Chk2pT68 was purchased from Millipore. Rabbit anti-ATM was purchased from Calbiochem. Rabbit anti-ATRpT1989 was a gift of Prof. Lee Zou.

Martínez T.F., Phillips J.W., Karanja K.K., Polaczek P., Wang C.M., Li B.C., Campbell J.L, & Dervan P.B. (2014). Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response. Nucleic Acids Research, 42(18), 11546-11559.

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Western Blot Analysis of DNA Damage Response

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Western blot analyses were performed with anti-γ-H2AX (Millipore), anti-H2AX (Sigma-Aldrich), anti-RAD9 (Santa Cruz or Bethyl), anti-RAD17, anti-RAD1, anti-TopBP1, anti-CLASPIN (Santa Cruz), anti-tubulin, anti-CHK1, anti-CHK1-pS345, and anti-CHK2-pT68 (Cell Signaling Technology) antibodies. The antibodies against the phospho-peptides (pThr292: LQAHSpTPHPDC, pThr313: CAMETpTIGNEG, pSer326: CEGSRVLPSIpS) were raised by MBL (Medical and Biological Laboratories (MBL), Japan). HRP-conjugated antibodies were used as secondary antibodies, the ECL signal was incorporated using a Licor Odyssey Fc imager system, and the quantification was performed with the Image Studio software.

Wakida T., Ikura M., Kuriya K., Ito S., Shiroiwa Y., Habu T., Kawamoto T., Okumura K., Ikura T, & Furuya K. (2017). The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress. eLife, 6, e29953.

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DNA Damage Response Markers Analysis

Cited 1 time

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Anti-γH2AX (Millipore 05-636), anti-RAD51 (Millipore ABE257), and anti-53BP1 (Novus Biologicals NB100-304) antibodies were used for the immunofluorescence analysis. Anti-CHK1-pS345 (Cell Signalling Technology 2348), anti-XRCC4^{40 (link)}, anti-RAD51C (Novus Biologicals NB100-177), and anti-tubulin (Sigma, T4026) antibodies were used for the western blot analysis.

Matsuzaki K., Kumatoriya K., Tando M., Kometani T, & Shinohara M. (2022). Polyphenols from persimmon fruit attenuate acetaldehyde-induced DNA double-strand breaks by scavenging acetaldehyde. Scientific Reports, 12, 10300.

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Protein Extraction and Mitochondrial Fractionation

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Protein extraction and immunoblotting were performed as previously described [32 (link)]. Mitochondria-cytoplasm fractionation of isolated hepatocytes was executed as detailed [79 (link)]. Antibodies used for immunoblotting include anti-CDK1 (1:1000; ab18; Abcam), anti-CDK2 (1:200; sc-6248; Santa Cruz Biotechnology), anti-CHK1 (1:400; sc-8408; Santa Cruz Biotechnology), anti-CHK1 pS345 (1:500; 2348; Cell Signaling Technology), anti-COX IV (1:500; 4844; Cell Signaling Technology), anti-CYC (1:1000; 4280; Cell Signaling Technology), anti-p21^Cdkn1a (1:200; sc-6246; Santa Cruz Biotechnology), anti-p53 (1:2000; #9282; Cell Signaling Technology), anti-p53 pS15 (1:2000; #9284; Cell Signaling Technology), anti-TUBB (1:5000; MRB-435P, Covance Antibody Products) and anti-HSP90 (1:5000; 610418; BD Biosciences). Quantification of blots was done using ImageJ software [77 (link)].

Dewhurst M.R., Ow J.R., Zafer G., van Hul N.K., Wollmann H., Bisteau X., Brough D., Choi H, & Kaldis P. (2020). Loss of hepatocyte cell division leads to liver inflammation and fibrosis. PLoS Genetics, 16(11), e1009084.

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Western Blot Analysis of DNA Damage Signaling

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Proteins were separated by SDS-PAGE and transferred to PVDF membrane. The following antibodies were incubated with membrane: anti-UBQLN1(1:1000, HPA054143, Sigma), anti-RPA70 (1:1000, sc-28304, Santa Cruz), anti-ATR pT1989 (1:1000, GTX128145, GeneTex), anti-CHK1 pS345 (1:1000, 2348T, Cell Signaling Technology), anti-Flag (1:2000, F1804, Sigma Aldrich), anti-GFP (1:2500, ab290, abcam), anti-HA (1:2000, 66006-2-Ig, Proteintech), anti-mCherry (1:1000, 26765-1-AP, Proteintech), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech). And the secondary antibodies are HRP-conjugated anti-rabbit or anti-mouse (KPL, Inc).

Zhou H., Xie C., Xie Y., He Y., Chen Y., Zhang C., Zhang Y., Zhao Y, & Liu H. (2023). UBQLN1 deficiency mediates telomere shortening and IPF through interacting with RPA1. PLOS Genetics, 19(7), e1010856.

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Antibodies and Reagents for DNA Damage Response

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The antibodies used in the present work were as follows: anti‐Flag (F3165, Sigma‐Aldrich), anti‐Flag@M2 Affinity Gel(AZ220, Sigma), anti‐TIP60 (sc‐166323, Santa Cruz Biotechnology), anti‐SENP3 (ab124790, Abcam), anti‐DNA‐PKcs (ab32556, Abcam), anti‐DNA‐PKcs pT2609 (ab97611) and pS2056 (ab18192) (both Abcam), anti‐ATM (sc‐135663, Santa Cruz Biotechnology), anti‐ATM pS1981 (ab81292, Abcam), anti‐HA (H9658, Sigma), anti‐acetylated‐lysine antibody (9441s, Cell Signaling Technology), anti‐histone H4 (2592) and anti‐CHK2 pT68 (2661) (both Cell Signaling Technology), anti‐H4K16ac (13534s, Cell Signaling Technology), anti‐CHK1 (sc‐8408, Santa Cruz Biotechnology), anti‐CHK1 pS345 (2348s, Cell Signaling Technology), anti‐CHK2 (ab109413, Abcam), anti‐β‐actin (TA‐09, ZSGB‐BIO), anti‐γH2AX(05‐636, EMD Millipore), Alexa Fluor 488‐labelled Goat Anti‐Mouse IgG(H+L)(A‐21202, Invitrogen), Alexa Fluor 568‐labelled Goat Anti‐ Rabbit IgG(H+L) (A‐11036, Invitrogen). Cisplatin (PHR1624), etoposide (E1383), campathecin (C9911) and mitomycin C (M0503) were purchased from Sigma‐Aldrich. Annexin V, FITC Apoptosis Detection Kit (AD10) was purchased from DOjindo.

Han Y., Huang X., Cao X., Li Y., Gao L., Jia J., Li G., Guo H., Liu X., Zhao H., Guan H., Zhou P, & Gao S. (2022). SENP3‐mediated TIP60 deSUMOylation is required for DNA‐PKcs activity and DNA damage repair. MedComm, 3(2), e123.

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Whole Cell Protein Extraction and Analysis

Cited 2 times

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Whole cell extracts were obtained by lysing cells in 1% SDS and 10 mM Tris-HCl (pH 7.4) supplemented with protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail; ThermoFisher). Viscosity of the samples was reduced by brief sonication. To detect PAR, cell extracts were performed as described previously^{31 (link)}. To detect ATM, ATM-pS1981, DNA-PK and DNA-PK-pS2056, cell extracts were prepared as previously described^{22 (link)}. Proteins were separated by SDS-PAGE and immunoblotted with the following antibodies: anti-actin (MAB1501; Millipore), anti-ATM (ab32420; Abcam), anti-ATM-pS1981 (ab81292; Abcam), anti-cleaved caspase-3 (#9664; Cell Signaling), anti-caspase-9 (#9502; Cell Signaling), anti-Chk1 (sc84081; SantaCruz), anti-Chk1-pS345 (#2348; Cell Signaling), anti-DNA-PK (ab1832; Abcam), anti-DNA-PK-pS2056 (ab18192; Abcam), anti-HIF1α (NB100–449; Novus), anti-PAR (AM80–100UG; Millipore), anti-p53 (#48818; Cell Signaling), anti-p53-pS15 (#9284, Cell Signaling), anti-PARP-1 (#9542; Cell Signaling), anti-TOP1 (ab109374; Abcam), anti-Tubulin (T5168; Sigma-Aldrich), anti-V5 tag (46–0705; Invitrogen). Immunoblotting was revealed by chemiluminescence using ChemiDoc MP System (Bio-Rad). Quantification of protein levels was done with Image Lab software (version 4.1).

Mouly L., Mamouni K., Gence R., Cristini A., Cherier J., Castinel A., Legrand M., Favre G., Sordet O, & Monferran S. (2018). PARP-1-dependent RND1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin. Cell Death & Disease, 9(9), 931.

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