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P chk1

Manufactured by Cell Signaling Technology
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P-Chk1 is a phospho-specific antibody that recognizes the phosphorylated form of Chk1 (Checkpoint Kinase 1) at Ser345. Chk1 is a serine/threonine-protein kinase that plays a critical role in the cellular response to DNA damage and replication stress.

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Lab products found in correlation

P chk2, by Cell Signaling Technology (29 mentions) P atr, by Cell Signaling Technology (17 mentions) P atm, by Cell Signaling Technology (15 mentions) γh2ax, by Cell Signaling Technology (12 mentions) P h2ax, by Cell Signaling Technology (9 mentions) Cleaved parp, by Cell Signaling Technology (8 mentions) Cleaved caspase 3, by Cell Signaling Technology (7 mentions) Gapdh, by Santa Cruz Biotechnology (7 mentions) Flag tag (flag), by Merck Group (7 mentions) Caspase 3, by Cell Signaling Technology (6 mentions) β actin, by Merck Group (6 mentions) P akt, by Cell Signaling Technology (5 mentions) Gapdh, by Cell Signaling Technology (5 mentions) β actin, by Cell Signaling Technology (5 mentions) P cdc2, by Cell Signaling Technology (4 mentions) Caspase 8, by Cell Signaling Technology (4 mentions) Rad51, by Cell Signaling Technology (4 mentions) P brca1, by Cell Signaling Technology (4 mentions) Survivin, by Cell Signaling Technology (4 mentions) Caspase 9, by Cell Signaling Technology (4 mentions)

Close spelling variants of this product

P-Chk1-Ser317 P-Chk1-317 Anti-p-CHK1 P-Chk1 (Ser296) P-Chk1 Ser345 Chk1-S345-P Anti-P-Chk1-Ser317 P-CHK1 antibody P-Chk1-S317

77 protocols using p chk1

1

Western Blot Analysis of DNA Damage Signaling

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Whole cell and homogenized tumor tissues were lysed in NP-40 lysis buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl and 1% Nonidet P40, supplemented with Complete Protease Inhibitor Cocktail tablets and PhosStop phosphatase inhibitors (Roche, Indianapolis, IN)] and prepared as previously described (26 (link)). Total cellular proteins (40 μg) were separated on 12% or 15% SDS/PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon).. The membrane was then blocked for 1 hour at room temperature in 5% (w/v) non-fat milk in TBS-Tween-20 and probed overnight with primary antibodies followed by anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) in blocking buffer for 1 h. Membranes were subsequently incubated in Immobilon Western Blot Chemiluminescent HRP Substrate (Millipore) and developed on biomax XAR film (Kodak). Primary antibodies were purchased from Cell Signaling Technology: γH2AX (Ser139), p-Wee1 (Ser 642), Wee1, p-cdc2 (Tyr15), p-BRCA1 (Ser1524), p-Chk1 (Ser345), p-Chk1 (Ser317), Chk-1, phospho-Chk2 (Thr68), PP2A-C, PP2A-A, cleaved caspase-3 (Asp175), cleaved PARP (Asp214), p-histone H3 (Ser10), p-ATR (Ser428), and p-(Ser) 14-3-3 binding motif.
Chang K.E., Wei B.R., Madigan J.P., Hall M.D., Simpson R.M., Zhuang Z, & Gottesman M.M. (2014). The Protein Phosphatase 2A Inhibitor LB100 Sensitizes Ovarian Carcinoma Cells to Cisplatin-Mediated Cytotoxicity. Molecular cancer therapeutics, 14(1), 90-100.
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2

Comprehensive Immunoblotting Methodology

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Immunoblotting was performed as described previously (35 (link)). The following antibodies were used at a concentration of 0.1 to 0.5 μg/ml: anti-pMCM2 (3378-1; Epitomics Inc.), anti-MCM2 (sc-9839; Santa Cruz Biotechnology), anti–pPOL II (04-1571; Millipore), anti–POL II (05-952; Millipore), anti-Lamin B1 (ab16048; Abcam), anti-FANCD2 (sc-20022; Santa Cruz Biotechnology), anti-pCDC6 (ab76422; Abcam), anti-CDC6 (ab109315; Abcam), pChk1 (#2348; Cell Signaling Technology), anti-Chk1 (#2345; Cell Signaling Technology), anti-pCDK1 (#9111; Cell Signaling Technology), anti-CDK1 (sc-954; Santa Cruz Biotechnology), anti-gH2AX (#2577; Cell Signaling Technology), anti-PARP1 (#9542; Cell Signaling Technology), anti–cyclin B1 (sc-752; Santa Cruz Biotechnology), anti-CDC7 (sc-56274; Santa Cruz Biotechnology), anti-DBF4 (ab124707; Abcam), and anti-GAPDH (MAB374; Chemicon). Immunoblotted proteins were visualized by chemiluminescence.
Iwai K., Nambu T., Dairiki R., Ohori M., Yu J., Burke K., Gotou M., Yamamoto Y., Ebara S., Shibata S., Hibino R., Nishizawa S., Miyazaki T., Homma M., Oguro Y., Imada T., Cho N., Uchiyama N., Kogame A., Takeuchi T., Kurasawa O., Yamanaka K., Niu H, & Ohashi A. (2019). Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor. Science Advances, 5(5), eaav3660.
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3

Antibodies Used in Xenopus TOPBP1 Research

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We used the following commercially sourced antibodies in this work: Myc (Millipore Sigma #M4439), GST (Millipore Sigma #05-782), CHK1 (Santa Cruz Biotechnology #sc-8408), P-CHK1 (Cell Signaling Technology #2341S), and P-ATM (ATM phospho S1981 Antibody Rockland # 200-301-400S). We also used our own antibody against Xenopus TOPBP1, HU142, which has been described44 (link). The antibody against MRE11 was a kind gift of Howard Lindsay45 (link).
Montales K., Kim A., Ruis K, & Michael W.M. (2021). Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system. Scientific Reports, 11, 467.
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4

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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5

Immunoblotting Antibody Panel for DNA Damage

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For immunoblotting experiments, we utilized the following antibodies: −γH2AX(S139) (Cell Signaling #80312), H2AX (Sigma‐Aldrich # 07627), pRPA32(T21) (Abcam #ab109394), RPA32 (Cell Signaling #35869), −pATR(T1986) (Cell Signaling #58014 and #30632), ATR (Cell Signaling #13934), pCHK1(S345) (Cell Signaling #12302), CHK1 (Cell Signaling #2360), pATM(S1981) (Abcam #ab81292), ATM (Cell Signaling #2873), pCHK2(T68) (Cell Signaling #2197), CHK2 (Cell Signaling #6334), pDNA‐PK(S2056) (Abcam# ab18192), DNA‐PK (Abcam# ab32566), vinculin (Sigma‐Aldrich # V9131), histone H3 (Sigma‐Aldrich #07‐690), and SLFN11 (Santa‐Cruz #sc‐374339). All antibodies were diluted at 1:1,000 except SLFN11 1:2,000, tubulin 1:4,000, and vinculin 1:4,000, and all secondary antibodies were diluted at 1:4,000.
Schultz C.W., Zhang Y., Elmeskini R., Zimmermann A., Fu H., Murai Y., Wangsa D., Kumar S., Takahashi N., Atkinson D., Saha L.K., Lee C., Elenbaas B., Desai P., Sebastian R., Sharma A.K., Abel M., Schroeder B., Krishnamurthy M., Kumar R., Roper N., Aladjem M., Zenke F.T., Ohler Z.W., Pommier Y, & Thomas A. (2023). ATR inhibition augments the efficacy of lurbinectedin in small‐cell lung cancer. EMBO Molecular Medicine, 15(8), e17313.
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6

Western Blot Analysis of Skin Proteins

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All proteins were extracted with RIPA lysis buffer and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA). For mouse skin, the tissue was homogenized in liquid nitrogen and then lysed in RIPA lysis buffer. Equal amounts of protein were subjected to electrophoresis. Western blotting was performed as described previously (32 (link), 33 (link)). Antibodies used included COX-2, AKT, p53, p21, Chk1, Chk2, XPC, DDB1, DDB2, γH2AX, β-actin, GAPDH (Santa Cruz), SIRT6, p-AKT (serine 473), Cleaved-caspase3, p-AMPK, p-Chk1, p-Chk2 (Cell Signal), acetylated k382 p53 (ac-p53,Abcam), and ac-H3K9 (Sigma).
Ming M., Han W., Zhao B., Sundaresan N.R., Deng C.X., Gupta M, & He Y.Y. (2014). SIRT6 promotes COX-2 expression and acts as an oncogene in skin cancer. Cancer research, 74(20), 5925-5933.
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7

Comprehensive Antibody Validation for Cell Biology

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The antibodies used in this study included α-SMA (ab7817), SM22α (ab14106), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K36me2 (ab9049), H3K36me3 (ab9050), which were bought from Abcam. β-actin (AC026) and RAB7 (A12308) were got from ABclonal. H3K9me3 (GTX121677), MMP2 (GTX634832), MYH10 (GTX634160), MMP9 (GTX100458), PCNA (GTX100539) were purchased from GeneTex. P-H3 (sc-8656-R) was obtained from Santa Cruz. LAMP3 (AP1827A) was bought from Abgent. STX17 (HPA001204) was got from ATLAS. H3K4me2 (#9725), H3K4me3 (#9727), H3K36me1 (#14,111), p-AKT (#4060), AKT (#4685), p-FOXO3 (#9466), FOXO3A (#12,829), p-P38 (#4511), P38(#8690), p-CDC2 (#4539), p-Rb (#8516), Rb (#9313), p-CHK1(#2348), P-CHK2 (#2197), LC3A/B (#12,741), SQSTM1 (#88,588), p-AMPKα (#5831), AMPKα (#2535), p-mTOR (#5536), mTOR (#2983), LAMP1 (#9091), and COL1A1 (#91,144) were purchased from Cell Signaling Technology.
He Y., Yi X., Zhang Z., Luo H., Li R., Feng X., Fang Z.M., Zhu X.H., Cheng W., Jiang D.S., Zhao F, & Wei X. (2022). JIB-04, a histone demethylase Jumonji C domain inhibitor, regulates phenotypic switching of vascular smooth muscle cells. Clinical Epigenetics, 14, 101.
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8

Immunoblot Analysis of DNA Damage Response Proteins

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Clofarabine (CLO) was purchased from Selleck Chemicals (Houston, TX, USA). Melphalan (MEL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies for the immunoblot were as follows: anti-RRM1, -RRM2 (Abcam, Cambridge, MA, USA); anti-GAPDH, -caspase-8, -caspase-9, -caspase-3, -phosphorylated (p)-p53, -p21, -PUMA, -γ-H2A.X, -p-ATM, -ATM, -p-ATR, -ATR, -p-Chk1, -Chk1, -p-Chk2, -Chk2, -RAD51, -53BP1, -BRCA1 (Cell Signaling Technology, Danvers, MA, USA); anti-p53 (DO-1) (Santa Cruz Biotechnology, Dallas, TX, USA); anti-Noxa (Millipore/Merck, Darmstadt, Germany); and anti-BRCA2 (Bethyl Laboratories, Montgomery, TX, USA).
Sagawa M., Ohguchi H., Harada T., Samur M.K., Tai Y.T., Munshi N.C., Kizaki M., Hideshima T, & Anderson K.C. (2017). Ribonucleotide reductase large subunit (RRM1) as a novel therapeutic target in multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5225-5237.
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9

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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10

Cellular Lysis and Antibody Detection

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Cells were lysed in NET-N buffer (20 mM Tris [pH 7.6], 1 mM ethylenediaminetetraacetic acid, 1% NP40, 150 mM NaCl) supplemented with protease inhibitor cocktail tablets (Roche). The following primary antibodies were used in this study: γ-H2A.X antibody (Cat# 05–636, Millipore); 53BP1 (Cat# MAB3802, Millipore); pATR (Cat# GTX128145, GeneTex); pChk1 (Cat# 2344s, Cell Signaling Technology); α-tubulin (Cat# 2148, Cell Signaling Technology); RNASEH2B (Cat# HPA040084, Sigma); and RNASEH2A (Cat# NBP1–76981, Novus).
Wang C., Wang G., Feng X., Shepherd P., Zhang J., Tang M., Chen Z., Srivastava M., McLaughlin M.E., Navone N.M., Hart G.T, & Chen J. (2018). Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition. Oncogene, 38(14), 2451-2463.
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