Anti cdc25c

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-CDC25C is a laboratory reagent used to detect and study the CDC25C protein, a key regulator of cell cycle progression. It is designed for use in various research applications, including immunohistochemistry, Western blotting, and flow cytometry.

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Close spelling variants of this product

CDC25C (59 citations) Anti-phospho-CDC25C (2 citations) Anti-p-CDC25C-Ser216 (2 citations) Anti‐p‐CDC25C (2 citations)

12 protocols using anti cdc25c

Antibody Panel for Protein Expression Analysis

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The mouse monoclonal antibodies, namely anti-PR (sc-166169), anti-ERβ (sc-53494), anti-c-Jun (sc-74543), anti-p-c-Jun (sc-822), anti-JNK (sc-7345), anti-p-JNK (sc-6254), anti-C/EBPβ (sc-7962), and rat monoclonal antibody anti-ERα (sc-53493) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The rabbit polyclonal antibodies, including anti-phospho-cdc2 (Tyr15) (#9111), anti-cdc2 (#9112), anti-cleaved caspase 3 (Asp175) (#9661), anti-caspase 3 (#9662); rabbit monoclonal antibodies, including anti-phospho-cdc25C (Ser216) (#4901), anti-cdc25C (#4688), anti-phospho-histone H3 (Ser10) (#3377), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (#2118), anti-PR A/B (#3153), anti-FOXO1 (#2880), and anti-IGFBP1 (#31025) antibodies; and the anti-rabbit (#7074) and anti-mouse (#7076) IgG, horse radish peroxidase (HRP)-linked antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-Ki-67 (M7240) was purchased from Dako (Agilent; Santa Clara, CA, USA). All antibodies were used at the concentrations recommended by the manufacturers.

Yoriki K., Mori T., Aoyama K., Tarumi Y., Kataoka H., Kokabu T, & Kitawaki J. (2022). Genistein induces long-term expression of progesterone receptor regardless of estrogen receptor status and improves the prognosis of endometrial cancer patients. Scientific Reports, 12, 10303.

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Protein Expression and Phosphorylation Analysis

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Cells were collected and lysed with NP-40 lysis buffer with protease inhibitors. Lysates were analyzed for protein expression/phosphorylation as described previously [25 (link),27 (link)]. The following antibodies were used at the indicated dilutions; all antibodies were purchased from Cell Signaling (Danvers, MA, USA): anti-p-AKT-S473 (1:1000, #4060), anti-AKT (1:1000, #4685), anti-p-p44/42 MAPK (Erk1/2) -Thr202/Tyr204 (1:1000, #4370), anti- p44/42 MAPK (Erk1/2) (1:1000, #4695), anti-p-p38 MAPK-Thr180/Tyr182 (1:1000, #9211), anti-p38 MAPK (1:1000, #8690), anti-p-cdc2-Tyr15 (1:1000, #4539), anti-cdc2 (1:1000, #9116), anti-p-CDC25C-Ser216 (1:1000, #4901), anti-cdc25C(1:1000, #4688), anti-p-Histone H3-ser10 (1:1000, #53348).

Bi J., Dixit G., Zhang Y., Devor E.J., Losh H.A., Newtson A.M., Coleman K.L., Santillan D.A., Maretzky T., Thiel K.W, & Leslie K.K. (2021). Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy. Pharmaceuticals, 14(7), 682.

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Protein Extraction and Western Blot Analysis

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Cells and tissues were lysed with cold RIPA buffer (50 mM Tris-Cl, PH 8.0, 2 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 2% SDS, 10 mM NaF) supplemented with mixture of protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). For the tissue samples, sonication was additionally applied. The concentration of total protein was measured using BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL). For the soluble protein detection, the conditioned media was collected and centrifuged at 13,000 rpm for 20 min before collecting the supernatant. Proteins were resolved by SDS PAGE and electrotransferred to PVDF membrane. Primary antibodies used in immunoblot analyses are as follows: anti-FCN3 (R&D systems; AF-2367), anti-V5 (Invitrogen; R96025), anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126), anti-p21 Waf1/Cip1 (Cell Signaling Technology, Beverly, MA; 2947), anti-CDC25C (Cell Signaling Technology; 4688), anti-Cyclin B1 (Cell Signaling Technology; 4138), anti-Cyclin D1 (Cell Signaling Technology; 2926) anti-PARP (Cell signaling Technology; 9542), anti-HSPA5(Abcam; ab21685), anti-DDIT3 (Cell Signaling Technology; 2895), and anti-α-Tubulin (Sigma; SAB3501072). After applying appropriate secondary antibodies, proteins were detected by enhanced chemiluminescence detection kit (AbClon, Korea) and ChemiDoc™ Imaging system (Bio-Rad)

Jang H., Jun Y., Kim S., Kim E., Jung Y., Park B.J., Lee J., Kim J., Lee S, & Kim J. (2021). FCN3 functions as a tumor suppressor of lung adenocarcinoma through induction of endoplasmic reticulum stress. Cell Death & Disease, 12(4), 407.

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Immunoblot Analysis of TNNC1-V5 Expressing Cells

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TNNC1-V5-expressing cells were harvested and lysed using RIPA buffer as described previously (Jung et al., 2015a (link)). Primary antibodies used in immunoblot analyses are as follows: anti-V5 (Invitrogen), anti-p53 (Santa Cruz Biotechnology, USA), anti-p21 Waf1/Cip1 (Cell Signaling Technology, USA), anti-CDC25C (Cell Signaling Technology), anti-Cyclin B1 (Cell Signaling Technology), anti-α-tubulin (AbFrontier, Korea), and anti-γH2AX (Millipore, USA). Peroxidase-conjugated secondary antibodies were used for detection in combination with AbSignal Western Blotting Detection Reagent kit (Abclon, Korea) or enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech, USA) following the manufacturer’s protocols.

Kim S., Kim J., Jung Y., Jun Y., Jung Y., Lee H.Y., Keum J., Park B.J., Lee J., Kim J., Lee S, & Kim J. (2020). Characterization of TNNC1 as a Novel Tumor Suppressor of Lung Adenocarcinoma. Molecules and Cells, 43(7), 619-631.

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Western Blotting and Co-IP Analyses

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For western blotting and Co-IP analyses, cells pellets were lysed by using modified RIPA buffer (5 mM EDTA, 2 mM Na₃VO₄, 5 mM NaF and 1 mM PMSF) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). For western blotting analysis, SDS-PAGE was performed, and proteins were electroblotted onto PVDF membranes. Co-IP analyses were performed using 300 μg proteins from whole cells lysates. Samples were incubated overnight in IP buffer (250 mM NaCl, 15 mM MgCl₂, 40 mM Hepes, 60 mM glycerophosphate) additioned with protease inhibitors with primary antibody in presence of CNRr-activated sepharose 4B (Pharmacia), resolved by SDS-PAGE, transferred into PVDF membranes and labeled with anti-HIF-1α and/or anti-p300 primary antibodies. The primary antibodies used for Immunoblotting were purchased from: Novus Biologicals, (anti-HIF-1α), Millipore™ (anti-p300 and anti-Cyclin A), Santa Cruz (anti-Cdc25C, anti-Cyclin E and anti-β-Actin), Cell Signaling Technology® Inc (anti-Cyclin B1, anti-Cyclin D1, anti-Caspase 3 and anti-Caspase 9) and Oncogene Research (anti-p53). The secondary antibodies used for Immunoblotting were purchased from: GE Healthcare (anti-rabbit and anti-mouse) and Santa Cruz (anti-goat).

Borsi E., Perrone G., Terragna C., Martello M., Dico A.F., Solaini G., Baracca A., Sgarbi G., Pasquinelli G., Valente S., Zamagni E., Tacchetti P., Martinelli G, & Cavo M. (2014). Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma. Oncotarget, 5(7), 1779-1792.

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Western Blot Analysis of Cell Signaling

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Whole-cell lysate preparation and Western blot analysis were performed as described [23,24] . Phospho-specific anti–DNA-PKcs antibodies were described previously [23–25] . Antibodies used in this study include anti-Cdk1, anti–phospho-Cdk1 (pY15), anti-Cdc25C, anti-Wee1, anti-Myt1, anti–cyclin B1, anti–poly(ADP-ribose) polymerase 1 (PARP-1) (Cell Signaling Technology, Danvers, MA), anti–phospho-histone H3 (pH3), anti–cyclin A (Upstate), anti-Plk1 (Bethyl Laboratories, Montgomery, TX), and anti–β-actin (Sigma-Aldrich, St. Louis, MO) and were commercially available as indicated.

Lee K.J., Shang Z.F., Lin Y.F., Sun J., Morotomi-Yano K., Saha D, & Chen B.P. (2015). The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry. Neoplasia (New York, N.Y.), 17(4), 329-338.

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Antibody Characterization for PBK Phosphorylation

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The PBK and phospho-PBK (pT9) antibodies were purchased from Cell Signaling Technology (4942 and 4941). Rabbit polyclonal phospho-specific antibodies against PBK T24, S32, and S59 were generated and purified by AbMart. The peptides used for immunizing rabbits were SVLCS-pT-PTINI (T24), INIPA-pS-PFMQK (S32) and RGLSH-pS-PWAVK (S59). The corresponding non-phosphorylated peptides were also synthesized and used for antibody purification and blocking assays. Anti-Flag antibody was from Sigma. Anti-PLK1 was from BioLegend. Anti-β-actin, anti-Mps1/TTK, anti-cyclin E1, and anti-cyclin B antibodies were from Santa Cruz Biotechnology. Anti-aurora-A, anti-CDK2, anti-glutathione S-transferase (GST), anti-BUB1, and anti-BubR1 antibodies were from Bethyl Laboratories. Anti-phospho-S10 H3, anti-YAP, anti-phospho-S127 YAP, anti-phospho-S397 YAP, anti-vimentin, anti-E-cadherin, anti-N-cadherin, anti-CDC25C, anti-CDK4, anti-CDK5, anti-CDK6, anti-Cyclin A2, anti-Cyclin E2, anti-MAD2, anti-phospho-S795 Rb, anti-Wee1, and anti-phospho-S642 Wee1 antibodies were from Cell Signaling Technology. Anti-β-tubulin (Sigma) antibodies were used for immunofluorescence staining.

Stauffer S., Zeng Y., Zhou J., Chen X., Chen Y, & Dong J. (2017). CDK1-mediated Mitotic Phosphorylation of PBK is Involved in Cytokinesis and Inhibits its Oncogenic Activity. Cellular signalling, 39, 74-83.

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Western Blot Analysis of Protein Markers

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The total protein lysate was analyzed by SDS–PAGE, transferred to a nitrocellulose membrane, and immunostained with the following antibodies overnight at 4 °C: anti-vimentin (1:1000, AbCam Supplementary Table 1); antiCD44 (1:1000, Cell Signaling Technology, Beverly, MA, USA, Supplementary Table 1); anti-p-Akt^Ser473 (1:1000, Cell Signaling Technology, Supplementary Table 1); anti-CDC25C (1:1000, Cell Signaling Technology); anti-cyclin D1 (1:1000, Cell Signaling Technology, Supplementary Table 1); anti-N-cadherin (1:1000, EMD Millipore, Billerica, MA, USA Supplementary Table 1); anti-GAPDH (1:5000, Santa Cruz Biotechnology, Supplementary Table 1); and anti-p21 (1:250, Santa Cruz Biotechnology, Supplementary Table 1). Anti-human GAPDH (1:5000, Santa Cruz Biotechnology, Supplementary Table 1) was used as a loading control. The membranes were incubated with the appropriate HRP-conjugated IgG (anti-rabbit 1:3000, Cell Signaling Technology, or anti-mouse 1:10 000, Santa Cruz Biotechnology, Supplementary Table 1), and proteins were detected by ECL (Thermo Scientific, Rockford, IL, USA, Supplementary Table 1).

Boufraqech M., Zhang L., Jain M., Patel D., Ellis R., Xiong Y., He M., Nilubol N., Merino M.J, & Kebebew E. (2014). miR-145 suppresses thyroid cancer growth and metastasis and targets AKT3. Endocrine-related cancer, 21(4), 517-531.

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Western Blot Analysis of Cell Cycle Regulators

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Preparation of whole-cell lysates and Western blot analyses were performed as described [28 (link)]. The primary antibodies used were anti-CyclinB1 (1:1,000, rabbit polyclonal; Cell Signaling), anti-Cyclin B1 (1:1000, mouse monoclonal [V152]; Abcam), anti-CDK1 (1:1, 000, rabbit polyclonal; Cell Signaling), anti-pSer216-CDC25c (1:1,000, rabbit monoclonal; Cell Signaling), anti-CDC25c (1:1,000, rabbit monoclonal; Cell Signaling), anti-p-JNK (1:1,000, rabbit monoclonal; Cell Signaling), anti-JNK (1:1,000, rabbit polyclonal; Cell Signaling), anti-p-p38 MAPK (1:1,000, rabbit monoclonal; Cell Signaling), anti-p38 MAPK (1:1,000, rabbit monoclonal; Cell Signaling), anti-p-Bcl-x_L (1:1,000, rabbit polyclonal; Thermo Scientific), anti-Bcl-x_L (1:1,000, rabbit monoclonal; Cell Signaling), anti-Mcl1 (1:1,000, rabbit monoclonal; Cell Signaling), anti-XIAP (1:1,000, rabbit polyclonal; Cell Signaling) and anti-PARP (1:1,000, rabbit polyclonal; Cell Signaling). Blots were stripped and normalized by reprobing with anti-EF1α (1:2,000, mouse monoclonal; Millipore) and β-actin (1:20,000, mouse monoclonal; Sigma).

Rajasekaran D., Siddiq A., Willoughby J.L., Biagi J.M., Christadore L.M., Yunes S.A., Gredler R., Jariwala N., Robertson C.L., Akiel M.A., Shen X.N., Subler M.A., Windle J.J., Schaus S.E., Fisher P.B., Hansen U, & Sarkar D. (2015). Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model. Oncotarget, 6(28), 26266-26277.

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Signaling Protein Immunoblot Analysis

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The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA): anti-phospho-STAT3 (Tyr705; 1:1000), anti-cleaved caspase3 (1:1000), anti-phospho-Chk2 (Thr68; 1:1000), anti-Chk2 (1:1000), anti-phospho-cdc2 (Tyr15; 1:1000), anti-phospho-cdc2 (Thr161; 1:1000), anti-cdc2 (1:1000), anti-phospho-cdc25C (Thr48; 1:1000), anti-cdc25C (1:1000), and anti-cyclinB1 (1:1000). Anti-STAT3 (1:1000), anti-GAPDH (1:2000), and anti-ataxia telangiectasia and Rad3-related protein (ATR) (1:1000) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-SOCS1 (1:1000) antibody was obtained from IBL (Fujioka, Gunma, Japan).

Natatsuka R., Takahashi T., Serada S., Fujimoto M., Ookawara T., Nishida T., Hara H., Nishigaki T., Harada E., Murakami T., Miyazaki Y., Makino T., Kurokawa Y., Yamasaki M., Miyata H., Nakajima K., Takiguchi S., Kishimoto T., Mori M., Doki Y, & Naka T. (2015). Gene therapy with SOCS1 for gastric cancer induces G2/M arrest and has an antitumour effect on peritoneal carcinomatosis. British Journal of Cancer, 113(3), 433-442.

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