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P chk1 s317

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P-Chk1-S317 is a polyclonal antibody that detects Chk1 phosphorylated at Serine 317. Chk1 is a key regulator of cell cycle checkpoint control and DNA damage response.

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Lab products found in correlation

Pchk2 t68, by Cell Signaling Technology (7 mentions) Pchk1 s345, by Cell Signaling Technology (3 mentions) Gapdh, by Cell Signaling Technology (3 mentions) Ab4731, by Abcam (2 mentions) C myc, by Merck Group (2 mentions) P t1989 atr, by GeneTex (2 mentions) Chk2 pt68, by Cell Signaling Technology (2 mentions) Gapdh hrp, by Abcam (2 mentions) Alpha tubulin, by Abcam (2 mentions) Ps824 kap1, by Fortis Life Sciences (2 mentions) Ps15 p53, by Cell Signaling Technology (2 mentions) Atm 2c1, by Santa Cruz Biotechnology (2 mentions) Ps1981 atm, by Cell Signaling Technology (2 mentions) P s966 smc1, by Fortis Life Sciences (2 mentions) Ps33 rpa, by Fortis Life Sciences (2 mentions) Ub pcna lys 164, by Cell Signaling Technology (2 mentions) Pcna hrp, by Santa Cruz Biotechnology (2 mentions) Hrp conjugated goat anti mouse rabbit igg, by Merck Group (2 mentions) Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (2 mentions) Protease inhibitor, by Merck Group (2 mentions)

Spelling variants of this product

P-Chk1 (77 citations) Chk1-S317 (3 citations)

15 protocols using p chk1 s317

1

Western Blot Analysis of DNA Damage Response

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Cells were extracted in 20mM Tris, pH7.5, 150mM NaCl, 0.1% NP-40, 10% glycerol (lysis buffer) supplemented with protease inhibitors (Sigma). Protein samples were separated by SDS–PAGE, and subsequently transferred onto polyvinylidene difluoride membranes. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:2000 unless otherwise indicated. The following antibodies were used: ATMIN (AB3271, Millipore, used at 1:5000), WRNIP1 (ab4731, AbCam, used at 1:10,000), RAD18 (S2980, Epitomics) pS1981-ATM (10H11.E12, Cell Signalling Technology), ATM (2C1, Santa Cruz), p-T1989-ATR (GTX128145, GeneTex), ATR (sc28901, Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), p-S317-Chk1 (2344, Cell Signalling Technology), Chk1 (sc8408, Santa Cruz), p-S966-Smc1 (A300-050A, Bethyl Laboratories), Smc1 (ab9262, AbCam), p53 (DO-1, Santa Cruz), pS15-p53 (Cell Signalling Technology), pT68-Chk2 (Cell Signalling Technology), Chk2 (Clone 7, Millipore), pS33-RPA (A300-246A, Bethyl Laboratories), RPA (sc56770, Santa Cruz), Ub-PCNA (Lys 164) (13439, Cell Signaling Technology), PCNA (2586, Cell Signaling Technology), PCNA-HRP (Clone PC10, sc56 Santa Cruz), His (A00186-100, Genscript), Flag M2 (Sigma), c-Myc (Sigma), β-actin (Sigma), alpha-tubulin (ab7291, AbCam), GAPDH-HRP (ab9385, Abcam) and HRP-conjugated goat anti-mouse/rabbit IgG (Sigma).
Kanu N., Zhang T., Burrell R.A., Chakraborty A., Cronshaw J., Da Costa C., Grönroos E., Pemberton H.N., Anderton E., Gonzalez L., Sabbioneda S., Ulrich H.D., Swanton C, & Behrens A. (2015). RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress. Oncogene, 35(30), 4009-4019.
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2

Western Blot Analysis of DNA Damage Response

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Cells were extracted in 20mM Tris, pH7.5, 150mM NaCl, 0.1% NP-40, 10% glycerol (lysis buffer) supplemented with protease inhibitors (Sigma). Protein samples were separated by SDS–PAGE, and subsequently transferred onto polyvinylidene difluoride membranes. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:2000 unless otherwise indicated. The following antibodies were used: ATMIN (AB3271, Millipore, used at 1:5000), WRNIP1 (ab4731, AbCam, used at 1:10,000), RAD18 (S2980, Epitomics) pS1981-ATM (10H11.E12, Cell Signalling Technology), ATM (2C1, Santa Cruz), p-T1989-ATR (GTX128145, GeneTex), ATR (sc28901, Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), p-S317-Chk1 (2344, Cell Signalling Technology), Chk1 (sc8408, Santa Cruz), p-S966-Smc1 (A300-050A, Bethyl Laboratories), Smc1 (ab9262, AbCam), p53 (DO-1, Santa Cruz), pS15-p53 (Cell Signalling Technology), pT68-Chk2 (Cell Signalling Technology), Chk2 (Clone 7, Millipore), pS33-RPA (A300-246A, Bethyl Laboratories), RPA (sc56770, Santa Cruz), Ub-PCNA (Lys 164) (13439, Cell Signaling Technology), PCNA (2586, Cell Signaling Technology), PCNA-HRP (Clone PC10, sc56 Santa Cruz), His (A00186-100, Genscript), Flag M2 (Sigma), c-Myc (Sigma), β-actin (Sigma), alpha-tubulin (ab7291, AbCam), GAPDH-HRP (ab9385, Abcam) and HRP-conjugated goat anti-mouse/rabbit IgG (Sigma).
Kanu N., Zhang T., Burrell R.A., Chakraborty A., Cronshaw J., Da Costa C., Grönroos E., Pemberton H.N., Anderton E., Gonzalez L., Sabbioneda S., Ulrich H.D., Swanton C, & Behrens A. (2015). RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress. Oncogene, 35(30), 4009-4019.
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3

Immunoblot Analysis of Chk1 Phosphorylation

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An immunoblot analysis was performed using a standard method with cell extracts that were prepared in lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 1 mM DTT, 0.5 mM PMSF, 50 μg/ml pepstatin A, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and a phosphatase inhibitor cocktail (Roche)). The following antibodies were used for immunoblotting: p-Chk1-S317 and p-Chk1-S345 (Cell Signaling Technology), actin and Chk1 (Santa Cruz), and Ino80 (Abcam, ab118787). The uncropped full-length images of all immunoblot gels are provided in Supplementary Figure 5.
Lee S.A., Lee H.S., Hur S.K., Kang S.W., Oh G.T., Lee D, & Kwon J. (2017). INO80 haploinsufficiency inhibits colon cancer tumorigenesis via replication stress-induced apoptosis. Oncotarget, 8(70), 115041-115053.
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4

Comprehensive Antibody Panel for DNA Damage Response

Cited 1 time
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We used the following rabbit antibodies: TOPBP1 (ab2402; Abcam), RAD51 (sc8349; Santa Cruz; ab63801; Abcam), pRAD51 S14 (Yata et al., 2012 (link)), cyclin A (NCL-CYCLINA; Leica), RPA70 (ab79398; Abcam), pRPA32 T21 (ab61065; Abcam), pRPA32 S4/8 (A300-245A; Bethyl), pRPA32 S33 (NB100-544; Novus), pCHK1 S317 (2344; Cell Signaling), pCHK2 T68 (2661; Cell Signaling), H3 (ab1791; Abcam), pH3 S10 (06–570; Millipore), BACH1 (sc-28738; Santa Cruz), pPTEN S380 (9551; Cell Signaling), pLRRK1 S1790 (H. Hanafusa, Nagoya University, Nagoya, Japan; Hanafusa et al., 2015 (link)); mouse antibodies: γH2AX (05–636; Millipore), cyclin A (sc-751; Santa Cruz), BrdU (RPN20AB; AP Biotech), RPA32 (ab2175; Abcam), BRCA1 (sc-6954; Santa Cruz), BRCA2 (OP95; Millipore), CHK1 (sc-8408; Santa Cruz), Importin-β (ab2811; Abcam), Lamin A/C (sc-7292; Santa Cruz), α-tubulin (sc-8035; Santa Cruz), PLK1 (05–844; Millipore; 331700; Zymed); and goat antibody: γ-tubulin (sc-7396; Santa Cruz).
Moudry P., Watanabe K., Wolanin K.M., Bartkova J., Wassing I.E., Watanabe S., Strauss R., Troelsgaard Pedersen R., Oestergaard V.H., Lisby M., Andújar-Sánchez M., Maya-Mendoza A., Esashi F., Lukas J, & Bartek J. (2016). TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity. The Journal of Cell Biology, 212(3), 281-288.
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5

DNA Damage Response Pathway Analysis

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pCHK1 (S317) (Cell Signaling, Danver, Massachusetts, catalog no.12302), pCHK2 (T68) (Cell Signaling catalog no. 2661), FANCD2 (Abcam, Cambridge, Massachusetts, catalog no. ab108928), BRCA1 (Oncogene, St. Louis, Missouri catalog no. OP92), γH2AX (S139) (Millipore, St. Louis, Missouri, catalog no. 05-636).
Spriggs C.C., Blanco L.Z., Maniar K.P, & Laimins L.A. (2019). Expression of HPV-Induced DNA Damage Repair Factors Correlates with CIN Progression. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists, 38(1), 1-10.
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6

Comprehensive DNA Damage Signaling Assay

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GFP (Abcam catalog no. ab6556), TRF1(Abcam catalog no. ab10579), GAPDH (Santa Cruz catalog no. sc-47724), OGG1 (Abcam catalog no. ab124741), Actin Cell Signaling catalog no. 3700), Lamin B1 Abcam catalog no. ab16048), Lamin A/C (Cell Signaling catalog no. 4777), γH2AX (Santa Cruz catalog no. sc-517348), 53BP1 (Novous catalog no. NB100-304), TRF2 (Novous catalog no. NB110-57130), MDM2 (Cell Signaling catalog no. 86934), p53(Santa Cruz catalog no. sc-126), p21 (Cell Signaling catalog no. 2947), p16 (Proteintech catalog no. 10883-1-AP), pRB S807/811 (Cell Signaling catalog no. 8516), pCHK2 T68(Cell Signaling catalog no. 2197), pCHK1 S317 (Cell Signaling catalog no. 12302), pATM S 1981 (Abcam catalog no. ab81292), CHK1 (Cell Signaling catalog no. 2360), H3K27me3 (Cell Signaling catalog no. 9733), H3K27Ac (Cell Signaling catalog no. 8173), LSD1 (Cell Signaling catalog no. 2184), cGAS (Cell Signaling catalog no. 66546), p62 (Cell Signaling catalog no. 39749).
Barnes R.P., de Rosa M., Thosar S.A., Detwiler A.C., Roginskaya V., Van Houten B., Bruchez M.P., Stewart-Ornstein J, & Opresko P.L. (2022). Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening. Nature Structural & Molecular Biology, 29(7), 639-652.
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7

Cell Cycle Analysis of DNA Damage Response

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Cells were pre-treated with caffeine, VE-822 or KU-60019 for 1 h followed by CX-5461 treatment. For nocodazole experiment, cells were pre-treated with CX-5461 for 2 h followed by nocodazole treatment. After drug treatments, cells were fixed in methanol and stored at −20°C till further processing. For cell-cycle analysis cells were spin down, washed twice in PBS and suspended in RNaseA containing propidium iodide (PI) solution and incubated for half an hour. Cells were run on BD FACScaliber flow cytometer (BD Biosciences) and final cell-cycle analysis was performed using FlowJo software (Tree Star). For phospho protein detection, fixed cells were incubated with pH3(S10)-FITC (Biolegend), pH3(S28) (Cell Signaling Technology), pCHK1(S317) (Cell Signaling Technology) or pCHK2(T68) (Cell Signaling Technology) and analyzed with flow cytometry.
Negi S.S, & Brown P. (2015). rRNA synthesis inhibitor, CX-5461, activates ATM/ATR pathway in acute lymphoblastic leukemia, arrests cells in G2 phase and induces apoptosis. Oncotarget, 6(20), 18094-18104.
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8

DNA Damage Response Protein Analysis

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A total lysate of 10–30μg protein was loaded on SDS-Gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight and subsequently, probed with secondary HRP-conjugated antibodies. Primary antibodies: GAPDH (Life Science H86504M), Beta-Actin (Cell Signaling 4967), Ku70 (Santa Cruz sc-1486), Ku80 (Santa Cruz sc-9034), XLF (Santa Cruz sc-393844), DNA Ligase IV (Santa Cruz sc-28232), H2A.X (Cell Signaling 2595), γ-H2A.X-S139 (Cell Signaling 9718), Chk1 (Santa Cruz sc-7898), p-Chk1-Ser345 (Santa Cruz sc-17922), p-Chk1-S317 (Cell Signaling 2344), Chk2 (Santa Cruz sc-9064), p-Chk2-Thr68 (Santa Cruz sc-16297).
Smolinska A., Swoboda J., Fendler W., Lerch M.M., Sendler M, & Moskwa P. (2020). MiR-502 is the first reported miRNA simultaneously targeting two components of the classical non-homologous end joining (C-NHEJ) in pancreatic cell lines. Heliyon, 6(1), e03187.
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9

Analyzing DNA Damage Response Proteins

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Antibodies against Chk1, pChk1 (S317), pChk1 (S345), pChk2 (T68), pChk2 (S516), γH2AX, pCdc2 (Y15), pCdc25c (S216), Cdc25a, phH3 (S10), PARP, cleaved PARP, 53BP1, cyclin A, cyclin B1, cyclin D, cyclinE, pCDK2 (T160) and RPA70 were purchased from Cell Signaling Technologies and pChk1 (S296) from Abcam.
Cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using a BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above
Rawlinson R, & Massey A.J. (2014). γH2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments. BMC Cancer, 14, 483.
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10

DNA Damage Response Proteins Analysis

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The antibodies against And-1, γ-H2AX, Chk1, p-Chk1-S317, p-Chk1-S345, RPA32, FLAG, BrdU and ATR were described previously (56 (link)); mouse monoclonal antibody CtIP was a gift of Dr Richard Baer (Columbia University, New York); antibodies against p-H2AX-S139, p-Chk1-S317, RAD50, MRE11, NBS1, BRCA1, Chk2, p-Chk2-T68 were from Cell Signaling; antibody against pRPA32-S4/S8 was from Bethyl Laboratories; antibodies against actin and cyclin A were from Santa Cruz Biotechnology.
Camptothecin was from Sigma and KU-55933 was from Tocris Bioscience.
Li Y., Li Z., Wu R., Han Z, & Zhu W. (2016). And-1 is required for homologous recombination repair by regulating DNA end resection. Nucleic Acids Research, 45(5), 2531-2545.
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