P cdc25c

Manufactured by Cell Signaling Technology

Sourced in United States

P-CDC25C is a primary antibody that recognizes the phosphorylated form of CDC25C, a protein phosphatase that plays a crucial role in cell cycle regulation. This antibody is a useful tool for researchers studying cell cycle progression and cell division processes.

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CDC25C (59 citations) P-CDC25c ser 216 (6 citations) Anti-p-CDC25C-Ser216 (2 citations) Anti‐p‐CDC25C (2 citations) P-216Ser CDC25C (2 citations)

17 protocols using p cdc25c

Digitoxin Induces Apoptosis Signaling

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A library of 78 natural compounds was obtained from Target Molecule Corp. Digitoxin (≥98% pure) was purchased from Baoji Herbest Bio-Tech Co., Ltd. MTT was supplied by Sigma-Aldrich (Merck KGaA). An Annexin-V-FITC/propidium iodide (PI) staining assay kit was obtained from Beyotime Institute of Biotechnology. The bicinchoninic protein assay kit (BCA) was purchased from Thermo Fisher Scientific Inc., while PI and 4′,6-dimidyl-2-phenylindole (DAPI) were purchased from Roche Diagnostics (Shanghai) Co. Ltd. Primary antibodies against cyclin-dependent kinase 1 (CDK1, #9116), cyclin B1 (#4138), phosphorylated (p)-CDK1 (Thr14) (#2543), p-histone H2AX (γH2AX, #9718), ATR (#2790), p-ATR (Ser428) (#2853), CHK2 (#6334), p-Chk2 (Thr68) (#2197), CDC25C (#4688), p-CDC25C (Thr48) (#12028), Bax (#5023), Bcl-2 (#15071), cytochrome c (#11940), caspase-9 (#9508) and-3 (#9662), cleaved-caspase-3 (#9579) and −9 (#20750), cleaved poly (ADP-ribose) polymerase (PARP) (#5625), β-actin (#4970) and the horse-radish peroxidase (HRP)-conjugated secondary antibodies (Anti-mouse IgG, #7076; Anti-rabbit IgG, #7074), Alexa Fluor 647-conjugated Anti-rabbit IgG (H+L) (#4414) were obtained from Cell Signaling Technology Inc., (dilution of primary antibodies, 1:1,000; dilution of secondary antibodies, 1:2,000).

Lei Y., Gan H., Huang Y., Chen Y., Chen L., Shan A., Zhao H., Wu M., Li X., Ma Q., Wang J., Zhang E., Zhang J., Li Y., Xue F, & Deng L. (2020). Digitoxin inhibits proliferation of multidrug-resistant HepG2 cells through G2/M cell cycle arrest and apoptosis. Oncology Letters, 20(4), 71.

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Immunoblotting and Immunocytochemistry Protocol

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Rat monoclonal anti-BrdU (#OBT0030; AbDSerotec, Raleigh, NC), rabbit polyclonal anti-cytoskeletal actin (β-actin) (#A300-491A; Bethyl Laboratories, Inc., Montgomery, TX) and mouse monoclonal anti-Ki67 (NCL-Ki67-MM1; Novocastra/Leica Microsystems, Inc., New Castle, UK), rabbit polyclonal to p85 fragment of PARP (#G734A, Promega, Madison, WI) and cyclin B (BD #61029; BD Biosciences, San Jose, CA) were procured from their respective manufacturers. Rabbit polyclonal to cleaved caspase-3 (#9661), p21^Cip1/WAF1 (#CS2947), Cdc25C (#CS4648), p-Cdc25C (#CS9528), Cdc2 (#CS9112) and p-Cdc2 (#CS9111) were purchased from Cell Signaling Technology, Inc., Danvers, MA. Antibodies for p53 (#SC-6243), p27^kip21 (#SC528), p19 (#SC-71810), cyclin E (#SC481), Cdk2 (#SC6248) and Cdk4 (#SC23896) were all from Santa Cruz Biotechnology Inc., CA. Peroxidase-conjugated secondary (H&L chain-specific) antibodies for Western blots, goat anti-mouse IgG (#401253) and goat anti-rabbit IgG (#401315) were purchased from Calbiochem-Novabiochem Corp., CA. Secondary antibodies for immunocytochemical analyses, Cy3-AffiniPure donkey anti-rat IgG (#712-165-153) and Cy3-AffiniPure goat anti-mouse IgG (#115-165-003) were procured from Jackson Immuno Research Laboratories, Inc., West Grove, PA.

Guha G., Lu W., Li S., Liang X., Kulesz-Martin M.F., Mahmud T., Indra A.K, & Ganguli-Indra G. (2015). Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells. PLoS ONE, 10(5), e0125322.

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Investigating DNA Damage Responses in HepG2 Cells

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Pine needle oil [dissolved in dimethyl sulfoxide (DMSO), ≤0.1%], RNase and propidium iodide (PI) solution were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-γ-H2AX (cat no. 05636; 1:1,000) antibody was purchased from EMD Millipore Billerica, MA, USA), ATM (cat no. 2873; 1:500), p-ATM (cat no. 13050; 1:1,000), p-p53 (S15; cat no. 9286; 1:1,000), p-CDC25C (S216; cat no. 4901; 1:500) p-CHK2 (T68; cat no. 2661; 1:1,000) and CHK2 (cat no. 2662; 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). CDC25C (cat no. sc-327; 1:1,000), β-actin (cat no. sc-47778; 1:1,000), anti-H2AX (cat no. sc-54606; 1:200), p53 (cat no. sc-98; 1:500) antibodies, goat anti-rabbit (cat no. sc-2030; 1:3,000) and anti-mouse secondary (cat no. sc-2031; 1:3,000) antibodies were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Protein extraction solution kit was purchased from Beijing SBS Genetech Co., Ltd., (Beijing, China). Dulbecco's modified Eagle's medium and bovine serum albumin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HepG2 cell line was purchased from the China Center for Type Culture Collection of Wuhan University (Wuhan, China). This cell line was originally thought to be a hepatocellular carcinoma, but is now known to be a hepatoblastoma cell line (21 (link)).

Qiu B., Jiang W., Qiu W., Mu W., Qin Y., Zhu Y., Zhang J., Wang Q., Liu D, & Qu Z. (2017). Pine needle oil induces G2/M arrest of HepG2 cells by activating the ATM pathway. Experimental and Therapeutic Medicine, 15(2), 1975-1981.

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Immunoblotting of Whole-Cell Protein Extracts

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Whole-cell protein extracts were prepared with ice-cold RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) with Complete Protease Inhibitor Cocktail (Roche Life Sciences, Indianapolis, IN). Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), α-tubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz). Uncropped versions of all blots are shown in Supplementary Figs. 8-12.

Wu C., Lyu J., Yang E.J., Liu Y., Zhang B, & Shim J.S. (2018). Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells. Nature Communications, 9, 3212.

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Immunoblotting Technique for Protein Analysis

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Immunoblotting was performed as described previously [39 (link)]. Briefly, whole cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) with a protease inhibitor cocktail (Pierce, Cramlington, UK). Lysates were separated by 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA), and incubated overnight at 4°C with the following primary antibodies: IRF8 (Santa Cruz Biotechnology, CA, USA), p21, p-cdc25C, poly (ADP-ribose) polymerase (PARP), p STAT1, STAT1, β-catenin, β-actin, Bcl-2, Histone H3 (Cell Signaling Technology, Danvers, MA, USA), active-β-catenin (Merck Millipore), CCND1, c-myc, GAPDH (Epitomics, Burlingame, CA, USA), followed by incubation with secondary antibody. The bands were visualized using ECL Plus Detection Reagents (RPN2132; GE Healthcare Life Science, Buckinghamshire, UK). All assays were performed three times, independently.

Luo X., Xiong X., Shao Q., Xiang T., Li L., Yin X., Li X., Tao Q, & Ren G. (2017). The tumor suppressor interferon regulatory factor 8 inhibits β-catenin signaling in breast cancers, but is frequently silenced by promoter methylation. Oncotarget, 8(30), 48875-48888.

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Apoptosis Signaling Pathway Analysis

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MDS-L and SKM1 cells (10⁷) were lysed with cell lysis buffer. Total cell extracts were fractionated on 10% sodium dodecyl sulfate polyacrylamide gels, electroblotted to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and reacted with primary antibodies including Bax, Bcl-2, cleaved caspase-9, p-p53, CDK1, p-CDC25C, Cyclin B1, Cylin D1, p38 MAPK, p-p38 MAPK, p44/42, p-p44/42, SAPK/JNK, p-SAPK/JNK, Akt, p-Akt, GSK-3α/β, p-GSK- 3α/β and GAPDH (purchased from Cell Signaling Technology). Immunoreactivity was determined using the enhanced chemiluminescence method (Pierce Chemical, Rockford, IL).

Xu F., He Q., Li X., Chang C.K., Wu L.Y., Zhang Z., Liu L., Shi W.H., Zhu Y., Zhao Y.S., Gu S.C., Fei C.M., Guo J., Wu D, & Zhou L. (2014). Rigosertib as a selective anti-tumor agent can ameliorate multiple dysregulated signaling transduction pathways in high-grade myelodysplastic syndrome. Scientific Reports, 4, 7310.

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Cell Cycle Regulation Antibody Panel

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Antibodies included p27 mouse monoclonal (BD Biosciences, Franklin Lakes, NJ, USA), rabbit polyclonal (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-lamin A (Abcam, Waltham, MA, USA), CDC25B (Santa Cruz Biotechnology), WEE1 (Santa Cruz Biotechnology), phospho-CDK1 (Y15; Cell Signaling Technologies, Danvers, MA, USA), DNAse IIβ (kind gift from Shigekazu Nagata, Osaka University), CDK1 (BD Biosciences), Ki67 (Vector Laboratories), CDC25B (Santa Cruz Biotechnology), CDC25C (Santa Crus Biotechnology), and pCDC25C (Cell Signaling Technologies).

Taylor A., Gu Y., Chang M.L., Yang W., Francisco S., Rowan S., Bejarano E., Pruitt S., Zhu L., Weiss G., Brennan L., Kantorow M, & Whitcomb E.A. (2023). Repurposing a Cyclin-Dependent Kinase 1 (CDK1) Mitotic Regulatory Network to Complete Terminal Differentiation in Lens Fiber Cells. Investigative Ophthalmology & Visual Science, 64(2), 6.

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Investigating Signaling Pathways in Cancer

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Dasatinib, BIBW2992, PD0325091 and Etoposide were purchased from Selleck Chemicals (Houston, TX) and prepared as 20 mM stock solutions in DMSO. Antibodies used included anti-YAP-TAZ, pLats, Lats1, Lats2, pMST1/2, pH2AX, MEK, pMEK(S217/221), ERK, pERK(T202/Y204), pRb, pS6, LC3A/B, ATM, pChk1, Chk1, pChk2, Chk2, pCDC25c, pATRIP, p27, p21, pp38, β-catenin, cyclin D1, survivin, p4EBP1, pSMAD2(S465–467), p70S6K, pCDC2, CDC6, γH2AX, ATR, pDNA-PK(S2056) and DNA-PK, (Cell Signaling Technology, Danvers, MA); BRAF and cyclin E (Santa Cruz, Dallas, TX) and ATM, Flag M2 and β-actin (Sigma-Aldrich, St. Louis, MO); pATM(S1981) (GeneTex, Irvine, CA). Predesigned siRNAs of the target genes were purchased from Dharmacon (Pittsburgh, PA) or Thermo Scientific (Rodckford, IL). pcDNA4-Chk1-Flag and pCMV5-TOPO-3Xflag-TAZ plasmids were purchased from Addgene (Cambridge, MA). ^WTBRAF plasmid was provided by Dr. W. Kolch (Systems Biology Irland and The Conway Institute, University College Dublin).

Peng S., Sen B., Mazumdar T., Byers L.A., Diao L., Wang J., Tong P., Giri U., Heymach J.V., Kadara H.N, & Johnson F.M. (2015). Dasatinib induces DNA damage and activates DNA repair pathways leading to senescence in non-small cell lung cancer cell lines with kinase-inactivating BRAF mutations. Oncotarget, 7(1), 565-579.

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Western Blot Analysis of Cell Signaling

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Bax (ab32503; 1:1000), Bcl-2 (ab32124; 1:2000), CDK1 (ab201008; 1:1000), Cyclin B1 (ab32053; 1:10000), Cyclin D1 (ab40754; 1:2000), CyclinD2 (ab207604; 1:1000), c-Myc (ab32072; 1:1000), Cytochrome c (ab133504; 1:10000), RAS (ab52939; 1:1000), P27 (ab32034; 1:5000), P21 (ab109520; 1:1000), and PDK1 (ab202468; 1:2000) were obtained from Abcam (Cambridge, U.K.). Antibodies for Bad (9268S; 1:1000), Caspase-3 (9662S; 1:1000), cleaved Caspase-3 (9664; 1:1000), Caspase-9 (32539; 1:1000), PARP (9532; 1:1000), CDC25C (4688S; 1:1000), p-CDC25C (4901; 1:1000), PCNA (13110; 1:1000), P53 (2527S; 1:1000), PTEN (9188; 1:1000), AKT (4685; 1:1000), p-AKT (4060; 1:2000), mTOR (2983; 1:1000), p-mTOR (5536; 1:1000), EIF4E (2067; 1:1000), SAPK/JNK (9252; 1:1000), p-SAPK/JNK (9255; 1:1000), MAPK/P38 (8690; 1:1000), and p-MAPK/P38 (4511; 1:1000) were obtained from Cell Signaling Technology (Beverly, MA, U.S.A.).

Chen X., Song J., Yuan D., Rao Q., Jiang K., Feng S., Zhu G., Yan C., Li Y, & Zhu J. (2021). Incaspitolide A extracted from Carpesium cernuum induces apoptosis in vitro via the PI3K/AKT pathway in benign prostatic hyperplasia. Bioscience Reports, 41(6), BSR20210477.

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Comprehensive Protein Extraction and Analysis

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For total protein extraction, cells were lysed by a lysis buffer containing protease inhibitor cocktail (Roche; USA) and PMSF (Roche; USA). To separate nuclear fraction, we used NE-PER nuclear and cytoplasmic extraction kit (Piece, Rockford, USA) according to the manufacturer’s instruction^{39 (link)}. Western blotting was then performed according to the routinely protocol including protein separation, membrane-transferring, block and primary and secondary antibody incubation. The primary antibodies were against β-actin (abcam; 1:5000; UK), GAPDH (abcam; 1:5000; UK), c-myc (proteintech; 1:1000; USA), RAD51 (abcam; 1:10000; UK), KU80 (abcam: 1:10000; UK), β-catenin (proteintech; 1:1000; USA) and phospho-γH2AX (Cell Signaling, 1:1000; USA), HMGB1 (abcam, 1:10000; UK) and p-chk1, p-chk2, p-cdc25C, ATRIP (Cell Signaling; 1:1000; USA), acetyl-H3 (Lys9) (absin, 1:5000; China), GFP (proteintech, 1:2000, USA). Finally, proteins were visualized with ECL and Kodak film without light.

Zhao Y., Yi J., Tao L., Huang G., Chu X., Song H, & Chen L. (2018). Wnt signaling induces radioresistance through upregulating HMGB1 in esophageal squamous cell carcinoma. Cell Death & Disease, 9(4), 433.

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