Chk1 g 4

Manufactured by Santa Cruz Biotechnology

Sourced in United States

CHK1 G-4 is a lab equipment product offered by Santa Cruz Biotechnology. It is a protein-based reagent designed for research purposes. The core function of CHK1 G-4 is to facilitate the study of the Checkpoint Kinase 1 (CHK1) protein, which plays a crucial role in cell cycle regulation and DNA damage response pathways.

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Anti-CHK1 (G-4) (9 citations)

8 protocols using chk1 g 4

Protein Extraction and Western Blot Analysis

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Total protein was extracted from OC cell lines with 1% NP40 lysis buffer containing 150 mM NaCl, 50 mM TrisHCl, 10% glycerol, 1 × Halt proteinase inhibitor cocktail, 5 mM NaF, and 1 mM NaOrthovanadate. Protein concentrations were estimated using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The proteins were separated on the NuPage 4–12% gel (Invitrogen, Carlsbad, CA) and the band was visualized using either Lumina Classico or Crescendo Western HRP substrate system (Millipore) depending on the signal intensities. Antibodies IKKε (Sigma, I4907), CHK1 G-4 (Santa Cruz, sc-8408), phospho-CHK1 Ser345 (Cell Signaling, #2348), phospho-CHK1 Ser296 (Cell Signaling, #2349), PARP-1 (Santa Cruz, sc-7150), cleaved PARP (Cell Signaling, #9541), phospho-H2A.X (Cell Signaling, #5438), H2A.X (Abcam, ab10475), Phospho-cdc2 (Tyr15) (Cell Signaling, #9111), cdc2 (Cell Signaling, #9112), p21 (Millipore, #05-345), p53-BP53.122 (Santa Cruz, sc-73566), and GAPDH (Millipore, MAB374), Topoisomerase II (Abcam, ab52934) were used in this study, and the secondary antibodies ECL anti-rabbit IgG HRP and ECL anti-mouse IgG HRP (GE Healthcare) were used at 1:5000 dilutions.

Kim M.K., Min D.J., Wright G., Goldlust I, & Annunziata C.M. (2014). Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21. Oncotarget, 5(24), 12788-12802.

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Western Blot Analysis of DNA Damage Signaling

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Total protein was extracted from OC cell lines with 1% NP40 lysis buffer containing 150 mM NaCl, 50 mM TrisHCl, 10% glycerol, 1 X Halt proteinase inhibitor cocktail, 5 mM NaF, and 1 mM NaOrthovanadate. Protein concentrations were estimated using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The proteins were separated on the NuPage 4–12% gel (Invitrogen, Carlsbad, CA) and the band was visualized using either Lumina Classico or Crescendo Western HRP substrate system (Millipore) depending on the signal intensities. Antibodies Chk1 G-4 (Santa Cruz, sc-8408), phospho-Chk1 Ser345 (Cell Signaling, #2348), phospho-Chk1 Ser296 (Cell Signaling, #2349), cleaved PARP (Cell Signaling, #9541), phospho-H2AX (Cell Signaling, #5438), Topoisomerase I (Abcam, ab3825), and GAPDH (Millipore, MAB374) were used in this study, and the secondary antibodies ECL anti-rabbit IgG HRP and ECL anti-mouse IgG HRP (GE Healthcare) were used at 1:5000 dilutions.

Kim M.K., James J, & Annunziata C.M. (2015). Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells. BMC Cancer, 15, 196.

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Immunoblotting Analysis of Cell Signaling Proteins

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Samples were lysed in 2x Laemelli sample buffer before separation by SDS-PAGE and transferring to PVDF membranes, blocked with 10% milk in TBST for 1 hour, and incubated with primary antibodies overnight. Antibodies: PTEN 6H2.1 (Millipore 04-035), DHODH (Protein Tech 14877-1-AP), vinculin (Sigma), pCHK1 (Cell Signaling 2341), CHK1 G-4 (Santa Cruz sc-8408), pCAD (Cell Signaling 12662), CAD (Cell Signaling 11933). HRP conjugated secondary antibodies were used to detect protein signals.

Mathur D., Stratikopoulos E., Ozturk S., Steinbach N., Pegno S., Schoenfeld S., Yong R., Murty V.V., Asara J.M., Cantley L, & Parsons R. (2017). PTEN regulates glutamine flux to pyrimidine synthesis and sensitivity to dihydroorotate dehydrogenase inhibition. Cancer discovery, 7(4), 380-390.

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DNA Damage Response Signaling Pathway

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Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate and 1 μg/ml leupeptin (Cell Signaling 9803S) with protease inhibitor PMSF (Cell Signaling 8553S). Western blots were performed with following antibodies: CHK1 (G-4; Santa Cruz Biotechnology SC-8408), Phospho-CHK1 [Ser345] (Cell Signaling Technology 2348S), phospho-histone H2A.X [S139] (EMD 05–636), phospho-RPA32 [S4/S8] (Bethyl A300–245A), phospho-KAP1 [S824] (Abcam ab84077), vinculin (Santa Cruz Biotechnology SC-25336), RAD51 (EMD PC-130), RAD51C (Novus) and Cyclin E (Santa Cruz Biotechnology SC-247).

Parmar K., Kochupurakkal B.S., Lazaro J.B., Wang Z.C., Palakurthi S., Kirschmeier P.T., Yang C., Sambel L.A., Farkkila A., Reznichenko E., Reavis H.D., Dunn C.E., Zou L., Do K.T., Konstantinopoulos P.A., Matulonis U.A., Liu J.F., D’Andrea A.D, & Shapiro G.I. (2019). The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(20), 6127-6140.

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Antibody Panel for DNA Damage Response

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The following antibodies were used: PCNA (FL261 and PC10; Santa Cruz Biotechnology), HA (3F10; Roche), Polη [15 (link)], REV1 [15 (link)], LaminB (C20; Santa Cruz Biotechnology), RAD18 (rabbit polyclonal), phospho-CHK1 (Ser345) (133D3; Cell Signaling), CHK1 (G4; Santa Cruz Biotechnology), actin (I19; Santacruz), γH2AX (Millipore), Ub (P4D1; Santa Cruz Biotechnology), and FLAG (M2; Sigma).

Kanao R., Masuda Y., Deguchi S., Yumoto-Sugimoto M., Hanaoka F, & Masutani C. (2015). Relevance of Simultaneous Mono-Ubiquitinations of Multiple Units of PCNA Homo-Trimers in DNA Damage Tolerance. PLoS ONE, 10(2), e0118775.

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Apoptosis Induction Pathway Modulation

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Artesunate, MG132, EB, and AO were purchased from Sigma-Aldrich Inc. Venetoclax (ABT-199) was purchased from Selleck Chemical. Cytarabine and Q-VD-OPh were purchased from MedChemExpress. Antibodies to PARP and caspase-8 were obtained from BD Biosciences. Antibodies to Bcl-2 (C-2), Actin (C-2), Mcl-1 (S-19), Mcl-1 (G-7), Bax (6A7) and Chk1 (G-4) were obtained from Santa Cruz Biotechnology, Inc. Antibodies to Mcl-1 (D35A5), Bim (C34C5), Bak (D4E4), Bax poly, Noxa (D8L7U), Cleaved Caspase-3 (Asp175), Phospho-Chk1 (Ser345) (133D3), and Phospho-Histone H2A.X (Ser139) (20E3) were obtained from Cell Signaling Technology, Inc. Antibodies to Noxa were obtained from Abcam, Inc. Bak (Ab-1) was obtained from Merck Millipore. NOXA(sc-37305), BIM(sc-29802), MCL1(sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA(s10708), BIM(s195011), MCL1(s8583)siRNA was purchased from Thermo Fisher Scientific.

Zhang J., Wang Y., Yin C., Gong P., Zhang Z., Zhao L., Waxman S, & Jing Y. (2022). Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis. Cell Death & Disease, 13(4), 379.

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DNA Damage Response Signaling Immunoblotting

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Immunoblot analysis was performed as described in detail previously [21 (link),22 (link)]. Primary antibodies against ATM (Bethyl Laboratories, Montgomery, TX, USA), phospho-ATM Ser1981 (Cell Signaling Technologies, Danvers, NJ, USA), ATR (Bethyl Laboratories), phospho-ATR Ser428 (Cell Signaling Technologies), p53 (Cell Signaling Technologies), phospho-p53 Ser15 (Cell Signaling Technologies), SMC1 (Bethyl Laboratories), phospho-SMC1 Ser966 (Bethyl Laboratories), Chk1 (G-4; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Chk1 Ser317 (Bethyl Laboratories), phospho-Chk1 Ser345 (Cell Signaling Technologies), Chk2 (H-300; Santa Cruz), phospho-Chk2 Thr68 (Cell Signaling Technologies), Wip1 (PPM1D; Bethyl Laboratories), Cdc25A (F-6; Santa Cruz) and GAPDH (Cell Signaling Technologies) were used for immunoblots.

Tatewaki N., Konishi T., Nakajima Y., Nishida M., Saito M., Eitsuka T., Sakamaki T., Ikekawa N, & Nishida H. (2016). Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase. PLoS ONE, 11(1), e0147570.

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Antibody Characterization for Cell Signaling

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The following commercial antibodies, and the indicated concentrations, were used in this study. C-Myc (#E0115; 1:1000), Chk1 (G-4) (#H2714; 1:1000) and GST (Z-5) (#K0713; 1:1000) were purchased from Santa Cruz Biotechnology. M2 anti Flag Mouse antibody (#SLBT7654; 1:5000), cdc27 (AF3.1) (1:1000) and Actin (#087M4850; 1:10,000) were purchased from Sigma. Cdh1 (#CC43-100UG; 1:500) was purchased from Calbiochem. Cyclin A2 (BF683) (#6; 1:1000), βTRCP1 (D13F10) (1:1000) and Phospho-Chk1Ser345 (133D3) (#15; 1:1000) were obtained from Cell Signaling. HA (#SJ254200; 1:1000) antibody was purchased from Biolegend. Plk1 (3F8) (#06050819; 1:500) was obtained from Enzo Life Sciences. HA antibody (HA.C5 #18181) (1:1000) was purchased from Abcam. Secondary antibodies for western blotting were purchased from LI-COR Biosciences. Anti-phospho-Histone H2AX (clone JBW301) (#2977883, 1:500) was purchased from EMD Millipore Corp. Alexa546-conjugated antibodies (#A11030) for immunofluorescence were purchased from Invitrogen.

Pal D., Torres A.E., Stromberg B.R., Messina A.L., Dickson A.S., De K., Willard B., Venere M, & Summers M.K. (2020). Chk1-mediated phosphorylation of Cdh1 promotes the SCFβTRCP-dependent degradation of Cdh1 during S-phase and efficient cell-cycle progression. Cell Death & Disease, 11(4), 298.

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