Anti cdk1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-CDK1 is a primary antibody that recognizes the Cyclin-Dependent Kinase 1 (CDK1) protein, also known as Cell Division Control Protein 1 (CDC2) or p34. CDK1 is a critical regulator of the cell cycle and plays a key role in the control of cell division.

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22 protocols using anti cdk1

Generation and Characterization of Anti-BEX4 Antibodies

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Mouse polyclonal antibodies against human BEX4 protein were generated in C57BL/6 mice. Briefly, purified GST-BEX4 protein was injected four times intraperitoneally. Rabbit polyclonal antibodies against C-terminal polypeptides 89–106 of human BEX4 protein were commercially generated (Youngin Frontier, Seoul, Korea). The other antibodies used in this study were as follows: anti-GFP, anti-PLK1, anti-CDK1, anti-aurora A, anti-cIAP-1, anti-cdc20 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-poly(ADP-ribose) polymerase-1 (PARP), anti-cleaved-caspase 9, cleaved-caspase 7, anti-active-caspase 3, anti-phospho-threonine (p-Thr) (Cell Signaling Technology, Danvers, MA, USA), anti-aurora B (BD Biosciences PharMingen, San Diego, CA, USA), anti-securin (PTTG1; Zymed, San Francisco, CA, USA), anti-Myc (Bethyl Laboratories, Montgomery, TX, USA), and Alexa Fluor (Invitrogen, Leek, The Netherlands). The following reagents were used: MG132, cycloheximide (CHX), dimethyl sulfoxide (DMSO) (AG Scientific, San Diego, CA, USA), nocodazole (Sigma-Aldrich), and PLK1 kinase inhibitor BI2536 (Axon Medchem, Groningen, Netherlands).

Lee J.K., Ha G.H., Kim H.S, & Lee C.W. (2018). Oncogenic potential of BEX4 is conferred by Polo-like kinase 1-mediated phosphorylation. Experimental & Molecular Medicine, 50(10), 138.

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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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Western Blot Analysis of Cell Signaling

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Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

WOO S.M., CHOI Y.K., KIM A.J., CHO S.G, & KO S.G. (2015). p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells. Molecular Medicine Reports, 13(2), 1091-1096.

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Immunoblotting Protein Detection Workflow

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All samples were mixed with NuPAGE sample loading buffer (Invitrogen) and heated at 95°C for 5 min. Antibodies used for western blotting were anti-FLAG M2 (mouse, Sigma), anti-ERK2 (rabbit, Santa Cruz), anti-α-tubulin (rat, AbD Serotec), anti-PKCβ2 C-terminal (rabbit, Santa Cruz), anti-CDK1 (mouse, Santa Cruz), anti-CDC25B (rabbit, Cell Signaling Technology), and anti-pT641 PKCα/β2 (rabbit, Cell Signaling Technology). All primary antibodies were used in 5% dry milk in PBS with 0.1% Tween 20 and incubated overnight at 4°C. Horseradish peroxidase-coupled secondary anti-mouse (Dako), anti-rabbit (Fisher), and anti-rat (Santa Cruz) antibodies were detected by enhanced chemiluminescence (ECL select, GE Healthcare) and Biomark X-ray (Carestream) or Super RX-N (Fujifilm) films.

Tischer T, & Schuh M. (2016). The Phosphatase Dusp7 Drives Meiotic Resumption and Chromosome Alignment in Mouse Oocytes. Cell reports, 17(5), 1426-1437.

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Quantifying CDK1 and LC3 in Treated HeLa Cells

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HeLa cells were grown on coverslips, treated or not with LLnL for 4 hours, and fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Immunostaining, using monoclonal anti-CDK1 (Santa Cruz) and polyclonal anti-LC3 (Novus Biologicals) antibodies, and counterstaining with DAPI to visualize the nuclei, was carried out according to standard protocols. Epifluorescence microscopy was performed using a Leica microscope.

Herrero-Ruiz J., Mora-Santos M., Giráldez S., Sáez C., Japón M.Á., Tortolero M, & Romero F. (2014). βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy. Oncotarget, 5(17), 7563-7574.

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Immunoblotting and EMSA to Characterize NF-κB Pathway

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Immunoblotting was performed using whole cell lysate as previously described^{37 (link)}. Primary antibodies used include: anti-p50 (sc7178, Santa Cruz Biotechnology), anti-GAPDH (sc-137179,), anti-CDK1 (sc-54), anti-HA (sc-7392), anti-actin (sc-1616). Unedited, full-length immunoblots are provided in Supplementary Information (Supplementary Figs. S7–Fig. S15). Several immunoblots were cut prior to antibody hybridization for reagent conservation. To identify the κB-site, the program TFSEARCH was used and a sequence with 86% homology with the canonical κB binding site was identified. For EMSA, nuclear fraction was isolated, a double-stranded oligonucleotide (5′-CGCTGAAGAGAATTCCCAAGGC-3′) (IDT) containing the decameric κB-consensus sequence was end labeled with [ɣ-³²P] ATP and used as a probe, and the assay performed as previously described^{13 (link)}. Supershift assays were performed using antibodies specific to p50 or BCL-3. Competition was performed by pre-incubating the mixture with cold specific and non-specific DNA probe.

Voce D.J., Bernal G.M., Cahill K.E., Wu L., Mansour N., Crawley C.D., Campbell P.A., Arina A., Weichselbaum R.R, & Yamini B. (2021). CDK1 is up-regulated by temozolomide in an NF-κB dependent manner in glioblastoma. Scientific Reports, 11, 5665.

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Western Blot Analysis of Apoptosis and Cell Cycle Markers

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Cells were lysed with radioimmunoprecipitation assay lysis and extraction buffers (Pioneer Technology, Xi'an, China), separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated at 4°C with the following primary antibodies: Anti-Bax (sc-20067, 1:1,000), anti-Bcl-2 (sc-509, 1:1,000), anti-Ku86 (sc-5280, 1:500), anti-Ku70 (sc-17789, 1:1,000), anti-Rad51 (sc-133089, 1:500), anti-cyclin B1 (sc-7393, 1:1,000), anti-CDK1 (sc-53219, 1:500), (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH (#5174, 1:3,000; Cell Signaling Technology, Inc., Danvers, MA, USA). Then incubated with secondary antibodies coupled with horseradish peroxidase at room temperature for 1.5 h, anti-mouse (#4410, 1:5,000) or anti-rabbit (#4414, 1:5,000) antibodies (Cell Signaling Technology, Inc.). The membranes were visualized using a chemiluminescence reagent (Millipore) and the ChemiDoc System (Bio-Rad, Hercules, CA, USA).

Fu S., Jin L., Gong T., Pan S., Zheng S., Zhang X., Yang T., Sun Y., Wang Y., Guo J., Hui B, & Zhang X. (2018). Effect of sinomenine hydrochloride on radiosensitivity of esophageal squamous cell carcinoma cells. Oncology Reports, 39(4), 1601-1608.

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Western Blot Analysis of Cellular Proteins

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To generate enough material for Western blotting, cells were seeded at 175,000/15 cm plates (for samples collected up to 6 days after treatment) or at a density of 300 cells/well in 6-well plates (for samples at 9 days after treatment). Cell extracts were prepared in RIPA buffer and analyzed by 10% SDS-PAGE. Western blots were probed with anti-PARP (1:750; cat. no. 9542, Cell Signaling Technology); anti-γ-tubulin (1:1,000; cat. no. T-5192, Sigma-Aldrich); anti-eEF1A, eEF2 or RPL13a (rabbit, 1:1,000; Cell Signaling Technology); anti-cyclin D1 (rabbit, 1:250–500; cat. no. SC-753, Santa Cruz Biotechnology® Inc., Dallas, TX); anti-nucleolin (mouse, 1:5,000; cat. no. SC8031, Santa Cruz Biotechnology); anti-Cdk1 (1:1,000; cat. no. 9116, Cell Signaling Technology), anti-phospho-Y15 Cdk1 (1:1,000; cat. no. 9111, Cell Signaling Technology); anti-p21 (1:500; Cell Signaling Technology); anti-γ-H2Ax (1:250; Abcam); HRP-conjugated secondary antibodies and SuperSignal West Pico Solutions (Thermo Fisher Scientific Inc., Waltham, MA).

Stickel S.A., Gomes N.P., Frederick B., Raben D, & Su T.T. (2015). Bouvardin is a Radiation Modulator with a Novel Mechanism of Action. Radiation research, 184(4), 392-403.

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Antibody Validation for Cell Signaling

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Anti-human CDK16 antibodies were purchased from Sigma and Proteintech. Anti-p53, anti-Myc, anti-GAPDH, anti-CDK1, anti-CDK2, anti-CyclinB1, anti-CyclinD1, and anti-MDM2 antibodies were obtained from Santa Cruz Biotechnology. Anti-human p27 was from Abgent, anti-Flag antibody was from Sigma, and anti-human p53-S315 P, Anti-p53-S33P, Anti-p53-S46P phospho-specific antibodies and anti-HA antibody were from Cell Signaling Technology. MG132 and Cycloheximide were purchased from Calbiochem and Sigma respectively.

Xie J., Li Y., Jiang K., Hu K., Zhang S., Dong X., Dai X., Liu L., Zhang T., Yang K., Huang K., Chen J., Shi S., Zhang Y., Wu G, & Xu S. (2018). CDK16 Phosphorylates and Degrades p53 to Promote Radioresistance and Predicts Prognosis in Lung Cancer. Theranostics, 8(3), 650-662.

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Cell Signaling Pathway Assays

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All reagents (e.g., arecoline hydrobromide, arecaidine hydrochloride, guvacine hydrochloride, etc.) were obtained from Sigma Chemical (St. Louis, MO), unless otherwise stated. BNAs were dissolved in sterile medium for cell treatment. As described in detail previously [17 (link)], DMEM, calf serum (CS), trypsin, and protein markers were purchased from Gibco-Invitrogen (Grand Island, NY). Antibodies specific for AMPK, phospho-AMPK, and cyclin B1 were obtained from Cell Signaling Technology (Billerica, MA, USA). All other antibodies (i.e., anti-CDK1, anti-CDK2, anti-p21, anti-p27, anti-p53, anti-actin, donkey anti-rabbit IgG-HRP, etc.) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Tian Z.H., Weng J.T., Shih L.J., Siao A.C., Chan T.Y., Tsuei Y.W., Kuo Y.C., Wang T.S, & Kao Y.H. (2018). Arecoline inhibits the growth of 3T3-L1 preadipocytes via AMP-activated protein kinase and reactive oxygen species pathways. PLoS ONE, 13(7), e0200508.

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