Cyclin d1 antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Cyclin D1 antibody is a lab equipment product designed to detect the Cyclin D1 protein. Cyclin D1 is a key regulator of the cell cycle and plays a crucial role in the progression of cells from the G1 phase to the S phase. The antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and quantify Cyclin D1 expression in cells and tissues.

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Cyclin D1 (991 citations) Anti-cyclin D1 antibody (22 citations) Antibody against cyclin D1 (4 citations) Rabbit anti-Cyclin D1 monoclonal antibody (3 citations) Antibody for cyclin D1 (2 citations) Cyclin D1 primary antibody (2 citations)

13 protocols using cyclin d1 antibody

Immunohistochemical Analysis of Cell Signaling

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Sections were treated with either p‐JAK2 antibody, p‐STAT3 antibody, cyclin D1 antibody (Cell Signaling Technology), CDK6 antibody (Cell Signaling Technology) with neuron‐specific nuclear protein (NeuN) antibody (Chemicon), glial fibrillary acidic protein (GFAP) antibody (Santa), or ionized calcium‐binding adapter molecule‐1 (Iba‐1) antibody (Cell Signaling Technology); either cyclin D1 antibody or CDK6 antibody with p‐STAT3 antibody at 4°C overnight. The sections were then incubated with a secondary antibody (Invitrogen) for 1 h at 22 ± 2°C. The nuclei were stained with DAPI.
For double staining of NeuN, GFAP, Iba‐1, cyclin D1, or CDK6 and TUNEL, slices were incubated with the primary antibodies against NeuN, GFAP, Iba‐1, cyclin D1, or CDK6, respectively, at 4°C overnight, with PBS used as a negative control. Following three washes in PBS, the sections were incubated with fluorescent‐labeled secondary antibodies. Sections were then stained using the TUNEL assay kit according to the manufacturer's instructions and counterstained with DAPI.

Zhao Y., Ding M., Yan F., Yin J., Shi W., Yang N., Zhao H., Fang Y., Huang Y., Zheng Y., Yang X., Li W., Ji X, & Luo Y. (2022). Inhibition of the JAK2/STAT3 pathway and cell cycle re‐entry contribute to the protective effect of remote ischemic pre‐conditioning of rat hindlimbs on cerebral ischemia/reperfusion injury. CNS Neuroscience & Therapeutics, 29(3), 866-877.

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MNNG-Induced Gastric Cancer Model

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N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was purchased from Tokyo Chemical Industry Co., Ltd. Ranitidine hydrochloride was purchased from Shanghai Hengshan Pharmaceutical Co., Ltd. Liquiritin, hesperidin, lobetyolin, curdione, costunolide and atractylenolide II were purchased from Chengdu Must Bio-Technology Co., Ltd. The H&E staining kit and prostaglandin E₂ (PGE₂) ELISA kit (cat. no. H099-1) were purchased from Nanjing Jiancheng Bioengineering Institute. Methanol and acetonitrile were purchased from EMD Millipore. Ethanol, acetic acid, xylene and formaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd. The gastrin (GAS), pepsin (PP), and somatostatin (SS) ELISA kits were purchased from Elabscience, Inc. Wnt1 was purchased from Abcam Co., Ltd. The β-catenin, glycogen synthase kinase-3β (GSK-3β) and cyclin D1 antibodies and the goat anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Cell Signaling Technology, Inc. The PCR primers for Wnt1, Wnt5a, β-catenin, GSK-3β, cyclin D1, MMP7 and GAPDH were produced by Nanjing Genscript Biological Technology Co., Ltd. TRIzol^® reagent, Maxima First Strand cDNA Synthesis Reverse transcription PCR kit and Maxima SYBR Green/ROX quantitative PCR kit were obtained from Thermo Fisher Scientific, Inc.

Yan Z., Xu T., Xu Y., Chen W., An Z, & Zhu F. (2021). Jianpiyiqi formula ameliorates chronic atrophic gastritis in rats by modulating the Wnt/β-catenin signaling pathway. Experimental and Therapeutic Medicine, 22(2), 878.

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Quantifying Cell Cycle Protein Levels

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Identification of the level change of cell cycle proteins (CDK2 and Cyclin D1) was investigated in this study. The effect of HAA₂₀₂₀ (500, 2500, 5000 nM), dinaciclib (5, 25, 50 nM), and their combination in MCF7 cells was tested following a previous report [23 (link)]. Briefly, MCF7 cells (1 × 10⁶ cells/well of a 6-well plate) were treated for 24 h. The lysis buffer was used to isolate total proteins, while the Bradford method was used to determine their concentration, which were then electrophoresed using a polyacrylamide gel and transferred to membrane. The membranes were incubated with CDK2 (# 2561, 1:1000, Cell signaling) and Cyclin D1 antibodies (# 2922, 1:1000, Cell signaling) for 2 h at room temperature and secondary antibody GAPDH (# 8884, 1:1000, Cell signaling) for 1 h. Visualization of the immunoreactivity was assessed using horseradish peroxidase (HRP)-conjugated secondary antibodies.

N. Abdalla A., Qattan A., H. Malki W., Shahid I., Akbar Hossain M, & Ahmed M. (2020). Significance of Targeting VEGFR-2 and Cyclin D1 in Luminal-A Breast Cancer. Molecules, 25(20), 4606.

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Western Blot Analysis of Cell Cycle Regulators

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Western blotting was performed as described previously [22 (link)]. Briefly, whole cell lysates were electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels and were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with primary antibodies against P21 antibody (1:2000; Cell Signaling Technology, Danvers, Massachusetts, USA), cyclin D1 antibody (1:2000; Cell Signaling Technology, Danvers, Massachusetts, USA), cyclin E1 (1:2000; Cell Signaling Technology, Danvers, Massachusetts, USA), CDK4 antibody (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), CDK6 antibody (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), KI67 antibody (1:3000; Servicebio, Wuhan, Hubei, China) and EZH2 antibody (1:2000; Cell Signaling Technology, Danvers, Massachusetts, USA). The membranes were then incubated with the corresponding secondary antibodies (Cell Signaling Technology, Boston, MA, USA) and visualized using an enhanced chemiluminescence kit (Vazyme, Nanjing, Jiangsu, China).

Fang P., Chen H., Ma Z., Han C., Yin W., Wang S., Zhu H., Xia W., Wang J., Xu L., Liu T, & Yin R. (2021). LncRNA LINC00525 suppresses p21 expression via mRNA decay and triplex‐mediated changes in chromatin structure in lung adenocarcinoma. Cancer Communications, 41(7), 596-614.

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Western Blot Analysis of Cell Signaling

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Western blot was performed routinely, and antibodies were used as follows. PTEN (D4.3) mAb (Cat # 9188S), Phospho-AKT (S473) (D9E) mAb (Cat # 4060S), TCF1/TCF7 (C63D9) mAb (Cat # 2203S), Phospho-mTOR (Ser2448) (D9C2) mAb (Cat # 5536T), mTOR (7C10) mAb (Cat # 2983T), Phospho-GSK-3-beta (S9) (D85E12) mAb (Cat # 5558S), β-catenin (D10A8) mAb (Cat #8480), caspase-3 (8G10) mAb (Cat #9665), cleaved caspase-3 (Asp175) (5A1E) mAb (Cat #9664), Bax (D2E11) mAb (Cat #5023), cyclin D1 antibody (Cat #2922), and actin antibody are all from Cell Signaling Technology. SLFN5 antibody (Cat #HAP017760) was from Sigma-Aldrich. The secondary antibodies, anti-rabbit or anti-mouse, were obtained from Beyotime (Shanghai, China). Membranes were reacted with enhanced chemiluminescence lighting reagent (ECL; Amersham Pharmacia, NJ, USA) and exposed to X-film in the dark.

Gu X., Zhou L., Chen L., Pan H., Zhao R., Guang W., Wan G., Zhang P., Liu D., Deng L.L., Zhao W, & Lu C. (2021). Human Schlafen 5 Inhibits Proliferation and Promotes Apoptosis in Lung Adenocarcinoma via the PTEN/PI3K/AKT/mTOR Pathway. BioMed Research International, 2021, 6628682.

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MTT Assay of LLL12 in DMSO

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LLL12 was provided by the Ohio State University College of Pharmacy and dissolved in DMSO. For the MTT assay (Research Products International, Mount Prospect, IL), the following antibodies were used: phospho-Stat3 (Y705) antibody, Stat3 antibody, cleaved caspase-3 (Asp175) antibody, cyclin D1 antibody, survivin antibody, and biotinylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (14C10) rabbit antibody (Cell Signaling Technology, Beverly, MA).

Zuo M., Li C., Lin J, & Javle M. (2015). LLL12, a novel small inhibitor targeting STAT3 for hepatocellular carcinoma therapy. Oncotarget, 6(13), 10940-10949.

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Detecting Endogenous LEF-1 Isoforms in Colon and HEK 293 Cells

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Endogenous LEF-1 isoforms were identified in colon cells and HEK 293 cells using the LEF-1 antibody (Cell Signaling, C12A5). c-MYC and cyclin D1 expressions in colon cells were detected using c-MYC antibody (Cell Signaling, D84C12) and cyclin D1 antibody (Cell Signaling, 92G2). GAPDH antibody was from Santa Cruz (sc-32233). Approximately 20–30 μg of cell lysates were analyzed in Western blots. Following SDS gel electrophoresis, the protein were transferred to PVDF filters (Millipore), immunoblotted and detected by specific antibodies and ECL plus reagents from GE HealthCare.

Zhang Z., Kim K., Li X., Moreno M., Sharp T., Goodheart M.J., Safe S., Dupuy A.J, & Amendt B.A. (2014). MicroRNA-26b Represses Colon Cancer Cell Proliferation by Inhibiting Lymphoid Enhancer Factor 1 (LEF-1) Expression. Molecular cancer therapeutics, 13(7), 1942-1951.

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Protein Expression Profiling by Western Blot

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Total proteins were extracted and subjected to SDS-PAGE. SETD2 antibody (Invitrogen), Cyclin D1 antibody (Cell Signaling Technology), Cyclin E1 antibody (Abcam), and H3K36me3 antibody (Cell Signaling Technology), phos-ERK antibody (Santa Cruz Biotechnology), ERK (Santa Cruz Biotechnology), phos-JAK2 (Santa Cruz Biotechnology), JAK2 (Santa Cruz Biotechnology), phos-STAT3 (Abcam) and STAT3 (Abcam) were used at a dilution of 1:2,000. H3 (Cell Signaling Technology, 1:2,000) and GAPDH (Santa Cruz Biotechnology, 1:2,000) were used as loading controls.

Zhou Y., Zheng X., Xu B., Deng H., Chen L, & Jiang J. (2020). Histone methyltransferase SETD2 inhibits tumor growth via suppressing CXCL1-mediated activation of cell cycle in lung adenocarcinoma. Aging (Albany NY), 12(24), 25189-25206.

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Evaluating COX-2 and mPGES-1 Signaling

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UA was bought from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin solution (EDTA) were bought from Gibco (Invitrogen, Grand island, NY). COX-2 (catalog no. 160106) and mPGES-1 (catalog no. 160140) antibodies were bought from Cayman Chemicals (Ann Arbor, MI). Cyclin A2 antibody (catalog no. ab7956) was purchased from Abcam (Cambridge, MA). Cyclin D1 antibody (catalog no. 2978) and GAPDH antibody (catalog no. ab9485) were purchased from Cell Signaling Technology (Danvers, MA). The PGE₂ enzyme immunoassay kit (catalog no. 514010-96) was provided by Cayman Chemicals (Ann Arbor, MI). COX-2 inhibitor NS-398 (catalog no. s1772) was from Beyotime (Shanghai, China).

Li S., Sun Z., Zhang Y., Ruan Y., Chen Q., Gong W., Yu J., Xia W., He J.C., Huang S., Zhang A., Ding G, & Jia Z. (2016). COX-2/mPGES-1/PGE2 cascade activation mediates uric acid-induced mesangial cell proliferation. Oncotarget, 8(6), 10185-10198.

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Lipid Metabolism and Cell Signaling

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All cell culture reagents were purchased from Gibco BRL Technology. AA, EPA, DHA, propidium iodide (PI), and PUFA analytical standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). Water-soluble tetrazolium (WST) reagent was from Dojindo Laboratories (Kumamoto, Japan). The following were commercially obtained antibodies: PTEN antibody, Ki-67 antibody, Cyclin D1 antibody, anti-phospho-Akt antibody (Ser473), Akt antibody (Cell Signaling Technology Danvers, MA, USA); anti-β-actin antibody (Abcam, Cambridge, UK); anti- COX-2 antibody and PGE₂ (Cayman Chemical Co, Ann Arbor, MI, USA)

Pan J., Cheng L., Bi X., Zhang X., Liu S., Bai X., Li F, & Zhao A.Z. (2015). Elevation of ω-3 Polyunsaturated Fatty Acids Attenuates PTEN-deficiency Induced Endometrial Cancer Development through Regulation of COX-2 and PGE2 Production. Scientific Reports, 5, 14958.

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