Cyclin e

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Cyclin E is a cell cycle regulatory protein that plays a crucial role in the transition from the G1 phase to the S phase of the cell cycle. It is an essential component of the cell's machinery for DNA replication and cell division.

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Anti-cyclin E (21 citations) Mouse anti-Cyclin E (4 citations) Mouse anti-Cyclin E (#4129) (2 citations) Rabbit anti-cyclin E (2 citations)

123 protocols using cyclin e

Molecular Response to Taselisib and Letrozole

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Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKT^Ser473, AKT, phospho-PRAS40^Thr246, phospho-S6^Ser235/236, phospho-S6^Ser240/242, S6, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-ERα^Ser118, phospho-ERα^Ser167, cleaved PARP, p110α, phospho-p70S6K^Thr389, PR, cyclin E, phospho-mTOR^Ser2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).

Hoeflich K.P., Guan J., Edgar K.A., O'Brien C., Savage H., Wilson T.R., Neve R.M., Friedman L.S, & Wallin J.J. (2016). The PI3K inhibitor taselisib overcomes letrozole resistance in a breast cancer model expressing aromatase. Genes & Cancer, 7(3-4), 73-85.

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Oroxylin A Anti-inflammatory Signaling

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Oroxylin A was bought from Novartis Pharmaceuticals (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution. Primary antibodies against CDK2, CyclinE, p27, p-p65, p65, and COX-2 were obtained from Cell Signaling (Beverly, MA, USA). Primary antibodies against E-cad, N-cad, Vimentin, p-IκB, IκB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotechnology. All other reagents were purchased from Sigma-Aldrich (Louis, MO, USA).

Sun X., Chang X., Wang Y., Xu B, & Cao X. (2019). Oroxylin A Suppresses the Cell Proliferation, Migration, and EMT via NF-κB Signaling Pathway in Human Breast Cancer Cells. BioMed Research International, 2019, 9241769.

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Evaluation of RY10-4 Anti-cancer Effects

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RY10-4 (>95%) was prepared previously in our laboratory as described by Yuan et al. [5 (link)]. Sulforhodamine B (SRB) was purchased from J&K Scientific (Beijing, China). Dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) were purchased from Sigma Aldrich (St. Louis, MO, USA). Hoechst 33342, propidium iodide (PI), reactive oxygen species assay kit (DCFH-DA), RIPA lysis buffer, BCA protein assay kit, and BeyoECL plus chemiluminescence kit were obtained from Beyotime Inc. (Shanghai, China). Annexin V-FITC/PI apoptosis detection kits was purchased from KeyGEN BioTECH (Nanjing, China). Specific antibodies against Bcl-2, Bax, p53, cyclin E, CDK2, cleaved caspase3, actin, STAT3, p-STAT3, p21, GAPDH and corresponding secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Zhang X., Wang Y., Han S., Xiang H., Peng Y., Wu Y., Pan S., Zhang Y, & Ruan J. (2016). RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo. PLoS ONE, 11(3), e0151679.

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Immunoblot Analysis of Cell Signaling Pathways

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CD8⁺ T cells were lysed with RIPA buffer (50mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% Sodium deoxycholate, and 150mM NaCl) with addition of Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and protein concentration was determined using the Bio-Rad protein assay (Hercules, California, U.S.A.). Proteins were resuspended in LDS Sample Buffer, loaded (15 μg/lane) onto NuPAGE Bis-Tris gels, and transferred to polyvinylidene difluoride (PVDF) membranes using iBlot2 system (LifeTechnologies, Grand Island, New York, U.S.A.). Proteins were detected using rabbit anti-S6K, phospho-S6K, AMPKα, phospho-AMPKα, Acetyl-CoA carboxylase (ACC), phospho-ACC, cyclin E, cdk2, phospho-Rb (Ser780), tubulin, and anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, Massachusetts, U.S.A.). Primary antibodies were used at a 1:1,000 dilution whereas secondary antibody was used at a 1:50,000 dilution. PVDF membranes were developed using ECL Western Blotting Substrate (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and exposed to Amersham hyperfilm ECL (GE Healthcare Life Sciences, Piscataway, New Jersey, U.S.A.)

Lee P.H., Yamada T., Park C.S., Shen Y., Puppi M, & Lacorazza H.D. (2015). G0S2 modulates homeostatic proliferation of naïve CD8+ T cells and inhibits oxidative phosphorylation in mitochondria. Immunology and cell biology, 93(7), 605-615.

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Notopterol-mediated Regulation of Cellular Signaling

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Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT, USA). An FITC Annexin V kit was obtained from Elabscience Biotechnology (Houston, TX, USA). Antibodies specific to ERK1/2, p-c-Jun, p-p65 (p-NF-κB), p-Akt, Cyclin E, CDK4, N-cadherin, Vimentin, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), whereas antibodies specific to p-ERK, p38, p-p38, STAT3, p-STAT3, Akt, c-Jun, MCM2, PCNA, Cylin D1, E2F, Fibronectin, ZEB-1, MT1-MMP, uPA, and Claudin-1 were purchased from Abclonal (Woburn, MA, USA). Antibodies for the detection of Ki67, Snail, and p65(NF-κB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellulose membrane and ECL reagent were supplied by GE Healthcare (Little Chalfont, UK). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA). Notopterol with purity of 98% was ordered from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Inthanon S., Dejkriengkraikul P, & Yodkeeree S. (2023). Notopterol Suppresses IL-17-Induced Proliferation and Invasion of A549 Lung Adenocarcinoma Cells via Modulation of STAT3, NF-κB, and AP-1 Activation. International Journal of Molecular Sciences, 24(20), 15057.

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Immunofluorescence Analysis of Cell Cycle Regulators

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Cultured cells in eight-well chamber slides (Millicell EZ SLIDES) were fixed with cold methanol for 15 min at −20 °C. Then cells were incubated with the following primary antibodies at 4 °C overnight: β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926), and cyclin E (Cell Signaling, 4129). The cells were then incubated with appropriate fluorochrome-conjugated secondary antibodies as described above. Isotype-matched control antibodies were used as negative controls. Nuclei were counterstained with DAPI Staining Solution, and then images were captured using Olympus inverted fluorescence microscope (IX73). For quantitative analysis of mean fluorescence intensity, cells with positive signals in at least six random fields were measured by Olympus cellSens software.

Chang T.H., Shanti R.M., Liang Y., Zeng J., Shi S., Alawi F., Carrasco L., Zhang Q, & Le A.D. (2020). LGR5+ epithelial tumor stem-like cells generate a 3D-organoid model for ameloblastoma. Cell Death & Disease, 11(5), 338.

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Protein Expression Analysis Protocol

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Abs against phospho-ATM (Ser1981), ATM, phospho-Chk1 (Ser317), Chk1, phospho-Chk2 (Thr68), Chk2, phospho-p53 (Ser15), PTEN, phospho-CDK2 (Thr160), CDK2, Cyclin E, Cyclin B1, CDC2, phospho-CDC2 (Tyr15), cleaved PARP (Asp214), phospho-STAT3 (Ser727), STAT3, STAT5, caspase-3 and active caspase-3 (Asp175) were all obtained from Cell Signaling (Danvers, MA, USA). Abs recognizing p53 (EMD Millipore, Darmstadt, Germany), p21 (Santa Cruz, Dallas, Texas) and phospho-STAT5 (Ser726/731) (Sabbiotech, College Park, MD, USA) were obtained as indicated.

Chen L.M., Liu P.Y., Chen Y.A., Tseng H.Y., Shen P.C., Hwang P.A, & Hsu H.L. (2017). Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression. Scientific Reports, 7, 11864.

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Investigating Cell Signaling Pathways

Cited 1 time

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Phosphate-buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin were all bought from Gibco (Grand Island, NY, USA). HepG2 and LO2 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 medium including 1% penicillin-streptomycin and 10% FBS in a 37 °C incubator supplied with 5% CO₂. cytochrome C, Bax, Bcl-2, p53, p21, cleaved PARP, PARP, cleaved caspase-3, caspase-3, cleaved caspase-9, caspase-9, CDK2, cyclin A, cyclin B, cyclin E, p-JNK, JNK, p-Erk, Erk, p-p38, p38, p-Akt, Akt, p-mTOR, mTOR, p-FAK, FAK, p-PI3K, PI3K, LC3A/B, p62, GAPDH, β-actin, and all secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). DAPI, cyclosporin A (CsA, an inhibitor of permeability transition), N-acetyl-L-cysteine (NAC), ROS assay kit, LDH-release assay kit, cell cycle and apoptosis analysis kit, and mitochondrial membrane potential assay kit for JC-1 were bought from Beyotime Institute of Biotechnology (Shanghai, China). Smp24 and FITC-labeled Smp24 were obtained as previously described by us [9 (link)].

Nguyen T., Guo R., Chai J., Wu J., Liu J., Chen X., Abdel-Rahman M.A., Xia H, & Xu X. (2022). Smp24, a Scorpion-Venom Peptide, Exhibits Potent Antitumor Effects against Hepatoma HepG2 Cells via Multi-Mechanisms In Vivo and In Vitro. Toxins, 14(10), 717.

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Cell Cycle Regulation and Apoptosis Signaling Assay

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Wst-1 assay kit was purchased from Daeillab (Daeillab, Korea). LY294002 (PI3K/Akt inhibitor) and PD98059 (Erk inhibitor), L-Ascorbic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). BIO (GSK-3β inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as p-Akt, total-Akt, β-actin, Cyclin E, p-cdk2, p21 were obtained from Cell Signaling Technology (Beverly, USA). and p-GSK-3β, total-GSK-3β, p-Erk, total-Erk, Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse™ Muse™ Cell Cycle Kit (MCH100106) and Muse™ Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany). 3M™ Tegaderm (sterile barrier to external contaminants) was purchased from 3 M (3 M, USA).

Nam G.H., Jo K.J., Park Y.S., Kawk H.W., Yoo J.G., Jang J.D., Kang S.M., Kim S.Y, & Kim Y.M. (2019). Bacillus/Trapa japonica Fruit Extract Ferment Filtrate enhances human hair follicle dermal papilla cell proliferation via the Akt/ERK/GSK-3β signaling pathway. BMC Complementary and Alternative Medicine, 19, 104.

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Investigating STAT3 Signaling in Cancer Cells

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QRT-PCR and western blotting were performed as described previously [22 (link)]. The primer for miR155HG is F 5′-GAGTGCTGAAGGCTTGCTGT-3′, R 5′-TTGAACATCCCAGTGACCAG-3′, for β-actin is F 5′-TCACCCACACTGTGCCCA-TCTACGA-3′, R 5′- CAGCGGAACCGCTCATTGCCAATGG-3′. The antibodies for western blot analysis were: anti-ANXA2 (1:1000; Abcam, Cambridge, UK), anti-cell cycle-related proteins (cyclin E, cyclin D, CDK4, CDK6) (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Bax (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bcl-2 (1:500; Santa Cruz Biotechnology), anti-caspase 3 (1:1000; Abcam) and anti-β-actin (1:1000, Cell Signaling Technology). Cells were treated with EGF Recombinant Human Protein Solution (Thermo Fisher Scientific, Waltham, MA, USA) or the SH-4-54 inhibitor of STAT3 phosphorylation (Selleck, Shanghai, China) according to the manufacturer’s protocol.

Wu W., Yu T., Wu Y., Tian W., Zhang J, & Wang Y. (2019). The miR155HG/miR-185/ANXA2 loop contributes to glioblastoma growth and progression. Journal of Experimental & Clinical Cancer Research : CR, 38, 133.

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