P cdc2

Manufactured by Cell Signaling Technology

Sourced in United States

P-cdc2 is a lab equipment product that detects the phosphorylation of the cell division cycle 2 (cdc2) protein. Cdc2 is a key regulator of the cell cycle and its phosphorylation state is an important indicator of cell cycle progression.

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Lab products found in correlation

35 protocols using p cdc2

Analyzing Cell Signaling Pathways

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Axin2 (#2151, Cell signaling technology), c-Myc (#1472–1, Epitomics), β-actin (#A1978, Sigma), pACC (#11818, Cell signaling technology), Total ACC (#3676, Cell signaling technology), pAMPK (#2535, Cell signaling technology), Total AMPK (#2532, Cell signaling technology), GAPDH (#GTX627408, GeneTex), Plk1 (#sc-17783, Santa Cruz), cyclinD1 (#2978, Cell signaling technology), cyclinB1 (#4135, Cell signaling technology), p-Akt (#9272, Cell signaling technology), Total-Akt (#1081–1, Epitomics), p-P70S6K (#9234, Cell signaling technology), Total-P70S6K (#2708, Cell signaling technology), p-4E-BP1 (#2855, Cell signaling technology), Total-4E-BP1 (#GTX109162, GeneTex), p-CDC2 (#9111, Cell signaling technology), Total-CDC2 (#GTX108120, GeneTex). Compounds: GSK461364 was purchased from Cayman Chemical (Ann Arbor, MI, USA). BI2536 was from Achemblock (Burlingame, CA). DAPI and PI were from ThermoFisher (Waltham, MA).

Xie Y., Zhang W., Guo L., Kril L.M., Begley K.L., Sviripa V.M., Chen X., Liu X., Lee E.Y., He D., Wang C., Gao T., Liu X., Evers B.M., Watt D.S, & Liu C. (2021). Potent Synergistic Effect on c-Myc Driven Colorectal Cancers Using a Novel Indole-Substituted Quinoline with a Plk1 Inhibitor. Molecular cancer therapeutics, 20(10), 1893-1903.

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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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Protein Expression Analysis in Tissues

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The cells and tissues were washed with phosphate-buffered saline (PBS) and then lysed using RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, and 150 mM NaCl, pH 8.0) with protease inhibitor mixture (Roche, USA) and phosphatase inhibitors (Roche, USA) at freezing condition for 15 min. The protein levels were measured with BCA Protein Assay Reagent kit (Thermo Scientific, USA). A 10% SDS-polyacrylamide gel was used to separate tissue lysate aliquots containing 20 μg protein. These were subsequently moved to PVDF membranes (Millipore), and the membranes were consequently blocked for 2 hours with TBST buffer with 5% skim milk at 22°C, and incubated at 4°C with primary antibodies overnight. We then added peroxidase-conjugated secondary antibodies and performed ECL (Cell Signaling Technology, 12757) visualization. Band enumeration was conducted using densitometric analysis software (Bio-Rad). GAPDH expression was used as the internal standard to standardize expression of the supplementary proteins. The primary antibodies were as follows: anti-ADNP (1:1000, Proteintech, USA), anti-GAPDH, CDK4, CDK6, Cyclin D1, Cyclin B1, p-cdc-2, p-Rb, E2F1, p53, MDM2, AKT, p-AKT, and p21 (1:1000, Cell Signaling Technology, USA). The secondary antibodies were HRP-Goat-anti-Rabbit Ig G and HRP-Goat-anti-Mouse Ig G (1:1000, Cell Signaling Technology, USA).

Zhu S., Xu Z., Zeng Y., Long Y., Fan G., Ding Q., Wen Y., Cao J., Dai T., Han W, & Xie Y. (2020). ADNP Upregulation Promotes Bladder Cancer Cell Proliferation via the AKT Pathway. Frontiers in Oncology, 10, 491129.

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Comprehensive Antibody Validation for Cell Biology

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The antibodies used in this study included α-SMA (ab7817), SM22α (ab14106), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K36me2 (ab9049), H3K36me3 (ab9050), which were bought from Abcam. β-actin (AC026) and RAB7 (A12308) were got from ABclonal. H3K9me3 (GTX121677), MMP2 (GTX634832), MYH10 (GTX634160), MMP9 (GTX100458), PCNA (GTX100539) were purchased from GeneTex. P-H3 (sc-8656-R) was obtained from Santa Cruz. LAMP3 (AP1827A) was bought from Abgent. STX17 (HPA001204) was got from ATLAS. H3K4me2 (#9725), H3K4me3 (#9727), H3K36me1 (#14,111), p-AKT (#4060), AKT (#4685), p-FOXO3 (#9466), FOXO3A (#12,829), p-P38 (#4511), P38(#8690), p-CDC2 (#4539), p-Rb (#8516), Rb (#9313), p-CHK1(#2348), P-CHK2 (#2197), LC3A/B (#12,741), SQSTM1 (#88,588), p-AMPKα (#5831), AMPKα (#2535), p-mTOR (#5536), mTOR (#2983), LAMP1 (#9091), and COL1A1 (#91,144) were purchased from Cell Signaling Technology.

He Y., Yi X., Zhang Z., Luo H., Li R., Feng X., Fang Z.M., Zhu X.H., Cheng W., Jiang D.S., Zhao F, & Wei X. (2022). JIB-04, a histone demethylase Jumonji C domain inhibitor, regulates phenotypic switching of vascular smooth muscle cells. Clinical Epigenetics, 14, 101.

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Apoptosis and Cell Cycle Analysis with Reagents

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The Cell Counting Kit-8 reagent was purchased from Dojindo (Japan). The cell cycle detection kit and Annexin-FITC apoptosis detection kit were purchased from KeyGen Biotech (Nanjing, China). Romidepsin (FK228) was purchased from Selleck Chemicals (Houston, TX). Romidepsin was dissolved in DMSO for the following in vitro and in vivo experiments. The primary antibodies against cleaved-caspase3, cleaved-caspase9, cleaved-PARP, PARP, CyclinB1, p-cdc2, Ki67 and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Li P., Liu L., Dang X, & Tian X. (2020). Romidepsin Induces G2/M Phase Arrest and Apoptosis in Cholangiocarcinoma Cells. Technology in Cancer Research & Treatment, 19, 1533033820960754.

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Immunoblot Analysis of Cell Cycle Regulators

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Whole cell lysates were obtained after treatment of the cells with BI2536 (0, 1, 10, and 50 nM) for 24 h. The membrane was blocked with 5% skim milk and immunoblotted with the primary antibodies at 4° C overnight. The following antibodies were used: PLK1 (Millipore, 05844), p-PLK1 (Abcam, EPR2612), Cdc2 (Cell signaling, 9112), p-Cdc2 (Cell signaling, 9111S), Cdc20 (MBL, K0140-3), p-Cdc20 (Cell signaling, 80385), Cdc25 (MBL, K0200-1), p-Cdc25 (Cell signaling, 9528S), cyclin B (Cell signaling, 4135), p-cyclin B (Cell signaling, 4131S), and APC3 (MBL, K0141-3). Then, horseradish peroxidase-labeled secondary antibodies were added (Chemicon, Single Oak Drive, Temecula, CA, USA), and visualization was performed using enhanced chemiluminescence (T-Pro Biotechnology, New Taipei City, Taiwan).

Cheng C.Y., Liu C.J., Huang Y.C., Wu S.H., Fang H.W, & Chen Y.J. (2018). BI2536 induces mitotic catastrophe and radiosensitization in human oral cancer cells. Oncotarget, 9(30), 21231-21243.

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Ovarian Tissue Protein Analysis

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The total protein was extracted from ovarian tissue according to the manufacturer’s instructions, and the total protein concentration was determined using the bicinchoninic acid assay (BCA; Solarbio, China). In each group, 10 μL of samples were electrophoresed on a sodium dodecyl sulfate-10% polyacrylamide gel and then transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked in 5% defatted milk at room temperature for 2 h, and then incubated with the primary antibody against GADD45b (Santa Cruz, USA, 1:1000), CyclinB1 (Cell Signaling Technology, USA, 1:1000), CDC2 (Cell Signaling Technology, 1:1000), pCDC2 (Cell Signaling Technology, 1:1000) and β-actin antibody (Cell Signaling Technology, 1:1000) diluted in 1 × TBST containing 5% defatted milk overnight at 4 °C, and then with HRP labeled secondary antibody (Beyotime, China, 1:1000) diluted in 1 × TBST containing 5% defatted milk at room temperature for 2 h. The bands were visualised using an enhanced chemiluminescence detection system (PerkinElmer, USA) and exposed by ImageQuant LAS 4010 Control Software (GE, USA). Fluorescence intensity was quantitated using Image J (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD). The GADD45b, CyclinB1, CDC2 and pCDC2 expressions were normalized to β-actin expression.

Guo F., Xia T., Zhang Y., Ma X., Yan Z., Hao S., Han Y., Ma R., Zhou Y, & Du X. (2019). Menstrual blood derived mesenchymal stem cells combined with Bushen Tiaochong recipe improved chemotherapy-induced premature ovarian failure in mice by inhibiting GADD45b expression in the cell cycle pathway. Reproductive Biology and Endocrinology : RB&E, 17, 56.

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Western blot analysis of cell cycle proteins

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Whole-cell extracts were subjected to SDS-PAGE on a 4% to 15% gel (Bio-Rad) and then transferred proteins onto PVDF membranes. After blocking nonspecific binding for 1 hour using 1× TBS 1% Casein blocker (Bio-Rad), the membranes were probed with specific primary antibodies overnight at 4°C and then incubated with a secondary HRP-conjugated mAb (1:5,000, Cell Signaling Technology) for 1 hour at room temperature. Proteins were visualized using an ECL system. Antibodies against Aurora A (rabbit; 1:1,000); p53 (mouse; 1:1,000); p-PLK1 (rabbit; 1:1,000); PLK1 (rabbit; 1:1,000); CDC2, (mouse; 1:1,000); p-CDC2 (rabbit; 1:1,000); Myc (rabbit; 1:1,000); p21 (rabbit; 1:1,000); cleaved-PARP (rabbit; 1:1,000); and Bcl-2 (mouse; 1:1,000) were all purchased from Cell Signaling Technology.

Deng M., Xu-Monette Z.Y., Pham L.V., Wang X., Tzankov A., Fang X., Zhu F., Visco C., Bhagat G., Dybkaer K., Chiu A., Tam W., Zu Y., Hsi E.D., You H., Huh J., Ponzoni M., Ferreri A.J., Møller M.B., Parsons B.M., Hagemeister F., van Krieken J.H., Piris M.A., Winter J.N., Li Y., Xu B., Liu P, & Young K.H. (2020). Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents. Molecular cancer research : MCR, 19(2), 249-260.

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Immunoblotting and Immunostaining for Cell Signaling

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For immunoblotting, cells were lysed in HNTG (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA) buffer containing phosphatase inhibitors (1 mM sodium orthovanadate and 1 mM NaF) and proteinase inhibitor cocktail (Roche). Cell lysates were subjected to immunoblotting using antibodies to tubulin (Santa Cruz), pRbS780, pRbS807/811, Rb, cyclin D1, pH3(S10), pAkt(S473), Akt, pFoxO1/3a, p p38MAPK, p38MAPK, p21^Cip1, p27^Kip1, pCdc2, Skp2, Spry1, p42/44MAPK (Cell Signaling Technology), dpERK (Sigma). For immunostaining carotid artery sections were subjected for antigen retrieval in citrate buffer (pH=5.5, 10mM), and stained with antibodies against PCNA, Spry1, Spry2, Spry4, SMTN-B (Santa Cruz), Ki67 (Cell Marque).

Yang X., Gong Y., He Q., Licht J.D., Liaw L, & Friesel R.E. (2017). Loss of Spry1 attenuates vascular smooth muscle proliferation by impairing mitogen-mediated changes in cell cycle regulatory circuits. Journal of cellular biochemistry, 119(4), 3267-3279.

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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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