Rabbit anti phospho chk1

Manufactured by Cell Signaling Technology

Sourced in United Kingdom, United States

Rabbit anti–phospho-CHK1 is a primary antibody that specifically binds to the phosphorylated form of the CHK1 protein. CHK1 is a serine/threonine-protein kinase that plays a crucial role in the cellular response to DNA damage and replication stress.

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5 protocols using rabbit anti phospho chk1

Kinase Assay of STAT3 and Chk1

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Kinase assays were performed using recombinant full-length human GST-STAT3 (SignalChem C47-10H) together with recombinant full-length human His-Chk1 (SignalChem S54-54G), in kinase buffer (20 mM Hepes pH 7.4, 10 mM MgCl₂, 10 mM MgCl₂, 1 mM EGTA, 0.1 mM Na3VO4, 0.5 mM NaF, 50 mM β-glycerophosphatase, 50 mM DTT) in the presence of 50 μM ATP and 5 μCi [³²P]-ATP (EasyTide). The kinase reaction proceeded for 45 min at 30 °C, and was stopped by the addition of NuPAGE™ LDS Sample Buffer (4X) (Invitrogen NP0007), boiled for 10 min at 85°C, and analyzed by electrophoresis on 4-12% SDS-polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue (CBB), then fixed, dried and subjected to autoradiography. A non-radioactive Kinase Assay was performed without cold ATP. Kinase products were analyzed by electrophoresis on 4-12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes, which were further stained Ponceau S. Immunoblotting was analyzed with rabbit anti-phospho-STAT3 (Tyr705, Cell Signaling Technology Cat# 2577), rabbit anti-phospho-STAT3 (Ser727, Cell Signaling Technology Cat# 9134) and rabbit anti-phospho-Chk1 (Ser296, Cell Signaling Technology Cat# 2349). Images were quantified and analyzed using ImageJ software (ImageJ, RRID:SCR_003070).

Zhou L., Pei X., Zhang Y., Ning Y., Li L., Hu X., Chalasani S.L., Sharma K., Nkwocha J., Yu J., Bandyopadhyay D., Sebti S.M, & Grant S. (2022). Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells. Molecular cancer research : MCR, 20(3), 456-467.

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Western Blot Analysis of Cell Signaling

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Whole cell lysates for Western Blot were prepared with the SDS-sample buffer. Proteins were separated by SDS-PAGE and transferred onto the PVDF membrane (Millipore/Fisher Scientific, Pittsburg, PA), Darmstadt, Germany). Antibodies for immunoblotting from Cell Signaling Technology (Dancers, MA) would include rabbit anti-Bmal1 (#14020), rabbit anti-CLOCK (#5157), rabbit anti-phospho-p53 (Ser15) [#9284], rabbit anti-p21 Waf1/Cip1 (#2947), rabbit anti-Histone H2AX (#2595), rabbit anti-cleaved PARP (#9541), and rabbit anti-phospho-CHK1 (Ser 345) (#2348). Other antibodies include mouse anti-p53 (sc-126; Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit anti-Keratin 10 (Poly19054; Biolegend, San Diego, CA), mouse anti-phospho-Histone H2AX (Ser139) (05–636; Millipore/Fisher Scientific), and mouse anti-α-tubulin (T9026; Sigma-Aldrich, St. Louis, MO). HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology Inc. Chemiluminescence images were acquired using an Amersham Imager 600 from GE Healthcare Life Sciences (Pittsburgh, PA) or iBright FL1000 (Invitrogen-Life Technology/Fisher Scientific, Pittsburgh, PA). The level of target proteins was quantified by densitometry scanning with the ImageJ software and normalized to the amount of α-tubulin.

Sun Y., Wang P., Li H, & Dai J. (2018). BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes. Journal of cellular physiology, 233(12), 9563-9574.

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Western Blot Analysis Methodology

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Cell lysates were prepared as described, added to 5× Laemmli buffer, and heated at 95°C for 5 min. Samples were run on Tris-glycine or NuPAGE Tris-acetate gels (Invitrogen) and transferred to polyvinylidene difluoride membranes at 35 V for 2 h. Membranes were blocked in PBS–0.5% Tween–5% milk for 1 h and stained with primary antibodies (1:250–1:2,000) for 1 h or overnight, followed by staining with secondary antibodies (1:10,000) conjugated with HRP (Jackson ImmunoResearch) for 1 h. After incubation with ECL substrate (Pierce/Thermo Fisher Scientific), blots were exposed to film. ImageQuant 5.2 (Molecular Dynamics) was used for quantification. The following primary antibodies were used for immunostaining: mouse anti–β-actin (A2228; Sigma-Aldrich), mouse anti-MYC (9E10, sc-40; Santa Cruz Biotechnology), rabbit anti–histone H3 (9715; Cell Signaling Technology), rabbit anti–histone H3K4me1 (ab8895; Abcam), rabbit anti–histone H3K27Ac (ab4729; Abcam), anti–phospho-Chk2 (2661, rabbit; Cell Signaling Technology), rabbit anti–phospho-RPA32 (S4/S8, A300-245A; Bethyl Laboratories), rabbit anti–phospho-CHK1 (2341S; Cell Signaling Technology), rabbit anti-UVSSA (GTX106751; GeneTex), rabbit anti-UVSSA (NBP1-32598; Novus Biologicals), and mouse S9.6 (ENH001; KeraFAST). HRP-conjugated secondary goat antirabbit and goat antimouse antibodies were obtained from Jackson ImmunoResearch.

Sato M., Liebau R.C., Liu Z., Liu L., Rabadan R, & Gautier J. (2021). The UVSSA complex alleviates MYC-driven transcription stress. The Journal of Cell Biology, 220(2), e201807163.

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Extraction and Analysis of Cellular Proteins

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Medium and cells were harvested in PBS-containing 1 mM Na₃VO₄ and 1 mM NaF. Cells were pelleted before lysis in 50 mM Tris.HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate and 0.1% SDS. Samples were thawed on ice, centrifuged at 14,000 rpm for 20 min at 4 °C and supernatants quantified by BCA assay from Pierce (Leicestershire, UK). 30 μg total protein lysate was separated by reducing SDS-PAGE, transferred to PVDF (GE Healthcare, Bucks, UK) and blocked with 5% non-fat dry milk in TBS. The following primary antibodies were used: rabbit anti-HSP72 from Stressgen (Exeter, UK); rabbit anti-GAPDH, rabbit anti-ATR, rabbit anti-phospho-ATR (S428), rabbit anti-CHK1, rabbit anti-phospho-CHK1 (S345), rabbit anti-RAD51, rabbit anti-ATM, rabbit anti-phospho-ATM (S1981), rabbit anti-phospho-BRCA1 (S1524), rabbit anti-phospho-p53 (S15) and rabbit anti-phospho-H2Ax (S139) were purchased from Cell Signaling (MA, USA); rabbit anti-FANCA was purchased from Bethyl Laboratories (TX, USA). Secondary antibodies used were sheep anti-mouse IgG and donkey anti-rabbit IgG HRP from GE Healthcare (Buckinghamshire, UK). Chemiluminescent detection was carried out using immobilon western substrate from Millipore (East Midlands, UK). In vivo samples were processed using a Precellys®24 homogenizer from Bertin Technologies (Montigny, France).

McLaughlin M., Barker H.E., Khan A.A., Pedersen M., Dillon M., Mansfield D.C., Patel R., Kyula J.N., Bhide S.A., Newbold K.L., Nutting C.M, & Harrington K.J. (2017). HSP90 inhibition sensitizes head and neck cancer to platin-based chemoradiotherapy by modulation of the DNA damage response resulting in chromosomal fragmentation. BMC Cancer, 17, 86.

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Immunoblotting Analysis of Phospho-CHK1

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Cells were exposed to 10 µM VE-821 for 24 h before lysis in RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF, with protease and phosphatase inhibitors). Protein samples were resolved in 4% to 12% SDS-PAGE gels and electroblotted onto a PVDF membrane. The membrane was blocked in 5% milk and tris-buffered saline, 0.1% Tween-20 (TBST) for 1 h, incubated with primary antibody diluted in blocking solution overnight at 4°C, washed 3 times with TBST, incubated with secondary antibodies for 1 h at room temperature, and washed 3 times with TBST. For the detection of signals, SuperSignal West Femto (Thermo Fisher Scientific) was used. Membranes were imaged on an Image LAS400 device (GE Healthcare Life Sciences, Chicago, IL, USA).
Primary antibodies and corresponding dilutions used are as follow: rabbit anti–phospho-CHK1 (1:500; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:1000; Invitrogen, Carlsbad, CA, USA). Blots were detected using goat anti- rabbit and goat anti-mouse (1:2500, Thermo Fisher Scientific). ImageJ was used for signal quantification.

Galofré C., Gönül Geyik Ö., Asensio E., Wangsa D., Hirsch D., Parra C., Saez J., Mollà M., Yüce Z., Castells A., Ried T, & Camps J. (2020). Tetraploidy-Associated Genetic Heterogeneity Confers Chemo-Radiotherapy Resistance to Colorectal Cancer Cells. Cancers, 12(5), 1118.

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