Rabbit anti cyclin d1

Manufactured by Cell Signaling Technology

Sourced in United States, China

Rabbit anti-cyclin D1 is a primary antibody that specifically recognizes the cyclin D1 protein. Cyclin D1 is a key regulator of the cell cycle, playing a crucial role in the G1 to S phase transition. This antibody can be used for the detection and analysis of cyclin D1 in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.

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Cyclin D1 (991 citations) Anti-cyclin D1 (332 citations) Rabbit anti-Cyclin D1 monoclonal antibody (3 citations) Rabbit monoclonal anti-Cyclin D1 (3 citations) Rabbit anti-human Cyclin D1 (2 citations)

29 protocols using rabbit anti cyclin d1

Gastric Cancer Cell Line Characterization

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HGC-27 and AGS gastric cancer cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, MKN45 and GES-1 were gifts from Digestive Tumor Research Institute of Fujian Medical University. HGC-27, GES-1 and MKN45 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Cat.11875–093, Cat.10099–141, Gibco,Thermo Fisher Scientific, Shanghai, China) and 1% Penicillin/Streptomycin Solution (100×, Gibco, Thermo Fisher Scientific, Shanghai, China). The total protein lysates were blotted with following antibodies: mouse anti-Pin1 (1:3000) from professor Lu of Harvard Medical University^{27 (link)}. Mouse anti-Actin (1:3000,#HC201–02; TransGen Biotech, Beijing, China).The following antibodies were purchased from Cell Signaling Technology: rabbit anti-Cyclin D1 (#2978,1:1000), rabbit anti-Phospho-Akt(Ser473) (#9271, 1:1000); rabbit anti-β-Catenin(#8480, 1:1000); rabbit anti-Phospho-GSK3beta(Ser9)(#9336,1:1000); rabbit anti-c-Myc (#9402, 1:1000), rabbit anti-CyclinD1(#2922, 1:1000), rabbit anti-CyclinE(#20808, 1:1000). All-trans retinoic acid (ATRA) powder were purchased from Sigma, ATRA-releasing pellets were from Innovative Research of America. SPF BALB/c nude mice were raised in Laboratory Animal Center of Fujian Medical University. All of animal experiments were approved by Experimental Animal Ethics of Fujian Medical University.

Zhang Z.Z., Yu W.X., Zheng M., X.i.n.-.h.u.a.-.L.i.a.o., Wang J.C., Yang D.Y., Lu W.X., Wang L., Zhang S., Liu H.K., Zhou X.Z, & Lu K.P. (2019). Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/β-catenin Oncogenic pathways. Molecular carcinogenesis, 58(8), 1450-1464.

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Endothelial Cell Culture and Signaling

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BD Matrigel™ Basement Membrane Matrix, culture dishes, and plates were obtained from Corning Inc. (Corning, NY, USA). Concanavalin A (Con A), sodium dodecyl sulphate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endothelial Cell Medium (ECM) was obtained from ScienCell (Carlsbad, CA, USA). PD98059 and LY294002 were the products of Tocris Bioscience (Bristol, UK). The primary antibodies, such as rabbit anti-phospho-Akt (Ser473) (#4060S), rabbit anti-Akt (#9272S), rabbit anti-phospho-ERK1/2 (#4730S), rabbit anti-ERK1/2 (#4695S), rabbit anti-phospho-p38 (#4511S), rabbit anti-p38 (#8690S), mouse anti-p27 (#3686S), mouse anti-p21 (#2947S), rabbit anti-cyclin D1 (#55506S), and rabbit anti-cyclin E (#20808S) antibodies were from Cell Signalling Technology (Beverly, MA, USA). The polyvinylidene fluoride (PVDF) membranes were the products of Millipore (Billerica, MA, USA). EdU Apollo®488 In Vitro Imaging Kit was a product of RIBOBIO (Guangzhou, China).

Li J.Z., Zhou X.X., Wu W.Y., Qiang H.F., Xiao G.S., Wang Y, & Li G. (2021). Concanavalin A promotes angiogenesis and proliferation in endothelial cells through the Akt/ERK/Cyclin D1 axis. Pharmaceutical Biology, 60(1), 65-74.

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Cell culture and Western blot analysis

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Cell lines Huh-7 and PLC/PRF/5 were bought from Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM media (#12800017; GIBCO) supplemented with 10% fetal bovine serum (#10437-028; GIBCO) and 1.5 g/L of NaHCO₃. The protein samples were blotted with following antibodies: rabbit anti-Cyclin D1 (#2978 S; Cell Signaling Technology) 1:1000, mouse anti-CDK6 (#3136 S; Cell Signaling Technology) 1:1000, rabbit anti-Phospho-Akt(Thr308) (#9275 S; Cell Signaling Technology) 1:600, rabbit anti-Phospho-Akt(Ser473) (#9271 S; Cell Signaling Technology) 1:600, rabbit anti-c-Jun (60A8) (#9165 S; Cell Signaling Technology) 1:1000, rabbit anti-B-Raf (#sc-166; Santa Cruz Biotechnology) 1;400, rabbit anti-Cleaved Notch1 (Val1744) (#4147 S; Cell Signaling technology)1:300, rabbit anti-β-Catenin (#8480 S; Cell Signaling technology) 1:1000, rabbit anti-LC3B (#ab48394; abcam) 1:1000, mouse anti-GSTP1 (#3369 S; Cell Signaling technology) 1:1000, mouse anti-Actin (#HC201; TransGen Biotech) 1:3000. BALB/c nude mice were housed in laminar flow cabinets with free access to food and water in Laboratory Animal Center of Fujian Medical University. All of animal experiments were performed in accordance with the animal protocols and regulations approved by FJMU Experimental Animal Ethics Committee of Fujian Medical University.

Liao X.H., Zhang A.L., Zheng M., Li M.Q., Chen C.P., Xu H., Chu Q.S., Yang D., Lu W., Tsai T.F., Liu H., Zhou X.Z, & Lu K.P. (2017). Chemical or genetic Pin1 inhibition exerts potent anticancer activity against hepatocellular carcinoma by blocking multiple cancer-driving pathways. Scientific Reports, 7, 43639.

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Quantitative Immunoblotting for Cellular Signaling

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Samples were lysed in RIPA lysis buffer (Thermo Scientific) supplemented with a protease inhibitor (Roche). Protein concentrations were measured using the Bradford Assay (Bio-Rad). Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked in 5% nonfat milk and incubated overnight at 4°C in blocking buffer containing primary antibodies. Antibodies used for immunoblotting were: rabbit anti-LC3B (1:1000; Sigma), rabbit anti-p21 (1:1000; Cell Signaling), rabbit anti-p27 (1:1000; Cell Signaling), rabbit anti-cyclin D1 (1:1000; Cell Signaling), mouse anti-p62 (1:1000; Abcam), rabbit anti-ERα (1:1000; Cell Signaling), rabbit anti-Atg7 (1:1000; Cell Signaling), rabbit anti-Beclin1 (1:1000; Cell Signaling), rabbit anti-ILK1 (1:100; Cell Signaling), and rabbit anti-GAPDH (1:2000; Cell Signaling). After three washes with TBST, blots were probed with horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 45 min (1:5000; Cell Signaling). Following incubation with ECL Plus Western Blotting Detection System (GE Healthcare), blots were imaged using a FluorChemE Imager (Cell Biosciences). For each protein, densitometry analysis was carried out in Image J by dividing the total intensity of each band by that of GAPDH in the same sample.

Anlaş A.A, & Nelson C.M. (2020). Soft microenvironments induce chemoresistance by increasing autophagy downstream of integrin-linked kinase. Cancer research, 80(19), 4103-4113.

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Immunoblotting and Immunohistochemistry Assays

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The antibodies used included mouse anti-β-actin (#A5441; Sigma-Aldrich; 1:5000 for Western blot), mouse anti-HA (#AE008; ABclonal, Cambridge, MA, USA; 1:2000 for Western blot), rabbit anti-ubiquitin-like 3 (UBL3) (#A4028; Abclonal; 1:1000 for Western blot and 1:100 for immunohistochemistry (IHC)), rabbit anti-Ki67 (#ab15580; Abcam, Cambridge, MA, USA; 1:400 for IHC), rabbit anti-p27 (#sc-528; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000 for Western blot), rabbit anti-Cyclin D1 (#2922; Cell Signaling Technology, Beverly, MA, USA; 1:1000 for Western blot), and mouse anti-Cyclin E (#4129; Cell Signaling Technology; 1:1000 for Western blot).

Zhao X., Yongchun Z., Qian H., Sanhui G., Jie L., Hong Y., Yanfei Z., Guizhen W., Yunchao H, & Guangbiao Z. (2020). Identification of a potential tumor suppressor gene, UBL3, in non-small cell lung cancer. Cancer Biology & Medicine, 17(1), 76-87.

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Immunoblotting Antibodies and Reagents

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Antibodies for Western blot were as follows: mouse anti-EGFR and mouse anti-HER4 (Abcam, Cambridge, UK), rabbit anti-HER3, mouse anti-p85 PI3K, rabbit anti-phospho EGFR (Try1068), rabbit anti-HER2, rabbit anti-cyclinD1, mouse anti-CDK4, rabbit anti-phospho p44/42 MAPK (p- Erk1/2), rabbit anti-p44/42 MAPK (Erk1/2), rabbit anti-Akt (Cell signaling technology, Danvers, MA), rabbit anti-phospho HER2 and rabbit anti-phospho Akt (Ser473) (Sigma-Aldrich, MO, USA). Recombinant human EGF was purchased from R&D systems, MN, USA. Varlitinib was supplied by ASLAN Pharmaceuticals, Singapore. BKM-120 was purchased from Active Biochem, NJ, USA. These were dissolved as stock in 100% dimethyl sulfoxide (DMSO) and stored at −80°C until use.

Dokduang H., Jamnongkarn W., Promraksa B., Suksawat M., Padthaisong S., Thanee M., Phetcharaburanin J., Namwat N., Sangkhamanon S., Titapun A., Khuntikeo N., Klanrit P, & Loilome W. (2020). In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines. Drug Design, Development and Therapy, 14, 2319-2334.

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Western Blot and Immunofluorescence Analysis

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The primary antibodies used were rabbit anti-E-cadherin, rabbit anti-N-cadherin, mouse anti-β-catenin and rabbit anti-PKD1 (1/500 for western blot and 1/50 for immunofluorescence; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-actin (1/200 for western blot; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-cyclin D1 (1/1000 for western blot, Cell signaling technology, Denvers, MA). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG (1/2000; Dako, Glostrup, Denmark), rabbit anti-goat IgG (1/2000 Santa Cruz Biotechnology) and goat anti-mouse IgG (1/5000; Rockland, Gilbertsville, PA). The Alexa Fluor-conjugated secondary antibodies were Alexa-Fluor-594-conjugated donkey anti-rabbit IgG, Alexa-Fluor-488-conjugated donkey anti-mouse IgG conjugated (1/200; Invitrogen, Cergy-Pontoise, France). Actin was stained with Alexa-Fluor-488-conjugated phalloidin (1/50; Invitrogen, Cergy-Pontoise, France). The nucleus was stained with 4',6-diamidino-2-phenylindole DAPI (1/50000; Invitrogen, Cergy-Pontoise, France). Gö6976 and Gö6983 were purchased from Calbiochem (Darmstadt, Germany). All other biochemicals were from Sigma-Aldrich (St. Louis, MO).

Merzoug-Larabi M., Spasojevic C., Eymard M., Hugonin C., Auclair C, & Karam M. (2017). Protein kinase C inhibitor Gö6976 but not Gö6983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1. BMC Cancer, 17, 12.

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Western Blot Analysis of ERα and Cyclin D1

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Cells were washed three times in cold 1×PBS and collected with a lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Equal amounts of protein lysate were used for western blot analyses with the indicated antibodies, which included: rabbit anti-ERα antibody (Santa Cruz Biotechnology, #sc-542, Santa Cruz, CA), rabbit anti-Cyclin D1 (Cell Signaling Technology, #2978s, Danvers, MA), Anti-β-actin Ab (Sigma, #A5411), anti-ERβ Ab (Novus Biologicals), and HRP coupled goat anti-mouse and HRP coupled goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology). Images were collected using an ImageQuant LAS 4000 (GE HealthCare, Pittsburgh, PA).

Du C., Li Z., Wang S., Zhou Z., Wang J., Dong J, & Chen C. (2014). Tongshu Capsule Down-Regulates the Expression of Estrogen Receptor α and Suppresses Human Breast Cancer Cell Proliferation. PLoS ONE, 9(8), e104261.

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Autophagy and Cell Cycle Regulation Assay

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The primary antibodies used in this study included the rabbit anti-Atg5 antibody (Cell signaling, cat:12994), rabbit anti-LC-3 antibody (MBL, cat: PM036), rabbit anti-p62 antibody (Cell Signaling, cat:5114), rabbit anti-actin antibody (Abcam, cat: ab8227), rabbit anti-Beclin-1 antibody (Abcam. ab51031), rabbit anti-Cyclin-D1 (Cell Signaling, cat:2978), rabbit anti-Cyclin-A2 (Cell Signaling, cat:4656), rabbit anti-Cyclin-E1 (Cell Signaling, cat:4129), rabbit anti-Cyclin-B1 (Cell Signaling, cat:4138), rabbit anti-p53 (Cell Signaling, cat:2524), rabbit anti-p21 (Abcam, ab7960), rabbit anti-p27 (Abcam, ab7961), rabbit anti-Caspase 8 (Cell Signaling, cat:9496), rabbit anti-Caspase 9 (Cell Signaling, cat:9509), rabbit anti-Caspase 3 (Cell Signaling, cat:9662), rabbit anti-Cytochrom C (Cell Signaling, cat:4272), rabbit anti-COX IV (Cell Signaling, cat:4844).

Chen Y., Xu Z., Zeng Y., Liu J., Wang X, & Kang Y. (2021). Altered metabolism by autophagy defection affect liver regeneration. PLoS ONE, 16(4), e0250578.

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Immunodetection of HPV E7 and p16

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Mouse monoclonal anti-HPV E7 (clone NM2) and anti-p16 (clone 50.1) were purchased from Santa Cruz Biotechnology. Rabbit mAb anti-CDK4 (D9G3E) and Rabbit anti-Cyclin D1 were purchased from Cell signaling technology. The CDK4/6 inhibitor IV (CAS 359886-84-3) was from Santa Cruz biotechnology.

Garcia-Bates T.M., Kim E., Concha-Benavente F., Trivedi S., Mailliard R.B., Gambotto A, & Ferris R.L. (2016). Enhanced cytotoxic CD8 T cell priming using DC expressing HPV-16 E6/E7-p16INK4 fusion protein with sequenced anti-PD1. Journal of immunology (Baltimore, Md. : 1950), 196(6), 2870-2878.

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