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Anti cyclin d1 antibody

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The Anti-Cyclin D1 antibody is a laboratory reagent that recognizes and binds to the Cyclin D1 protein. Cyclin D1 is a key regulator of the cell cycle and is involved in the progression from the G1 phase to the S phase of the cell cycle. The Anti-Cyclin D1 antibody can be used in various laboratory applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the expression and localization of Cyclin D1 in biological samples.

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Lab products found in correlation

Protein g agarose, by Thermo Fisher Scientific (2 mentions) Protein a agarose, by Thermo Fisher Scientific (2 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (2 mentions) Sc 6248, by Santa Cruz Biotechnology (2 mentions) Anti vamp2 antibody, by Synaptic Systems (1 mentions) Az100, by Nikon (1 mentions) Mouse on mouse immunodetection kit, by Vector Laboratories (1 mentions) Hematoxylin solution, by Muto Pure Chemicals (1 mentions) Anti foxm1, by Cell Signaling Technology (1 mentions) Lsm780 uv, by Zeiss (1 mentions)

Close spelling variants of this product

Cyclin D1 (46 citations) Anti-cyclin D1 (8 citations)

6 protocols using anti cyclin d1 antibody

1

Histological Analysis of Cellular Markers

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Luxol fast blue (LFB) staining was performed according to the manufacturer’s protocol (LFB Solution; Muto Pure Chemicals, Tokyo, Japan). H&E staining was performed using hematoxylin solution (Muto Pure Chemicals) and 1% eosin in water (Wako Pure Chemicals). Immunofluorescence staining was performed using a Mouse-on-Mouse immunodetection kit (Vector Laboratories, Burlingame CA), anti-VAMP2 antibody (1:200, cl:69.1; Synaptic Systems, #104211, Goettingen, Germany), anti-cyclin D1 antibody (1:50, cl:SP4; Thermo Fisher Scientific KK, #RM-9104-SO, Yokohama, Japan), AlexaFluor488-conjugated donkey anti-rabbit IgG F(ab’)2 fragment (1:500; Jackson ImmunoResearch, West Grove, PA), and Rhodamine RedX-conjugated donkey anti-mouse IgG F(ab’)2 fragments (1:500, Jackson ImmunoResearch). Microscopic images were obtained using a Nikon AZ-100 (Tokyo, Japan) and a cooled CCD camera (SPOT RT3; Diagnostic Instruments, Sterling Heights, MI).
Tajika Y., Murakami T., Iijima K., Gotoh H., Takahashi-Ikezawa M., Ueno H., Yoshimoto Y, & Yorifuji H. (2017). A novel imaging method for correlating 2D light microscopic data and 3D volume data based on block-face imaging. Scientific Reports, 7, 3645.
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2

FOXM1-Cyclin D1 Colocalization in Cd-Treated Cells

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The PLA kit (DUO92101-1KT, Sigma-Aldrich, St. Louis, MO) was used to determine the association between FOXM1 and cyclin D1 in Cd-treated ht-UtLM cells by colocalization of these two proteins following the manufacturer’s protocol. Briefly, the cells were seeded on 3.5 cm glass bottom plate at density of 30,000 cells/plate. After ON culture, cells were treated with Cd at 0, 0.1 or 10 µM with or without PD for 24 h, fixed with 4% of paraformaldehyde for 20 min, and permeabilized with 0.1% triton X-100 for 30 min. The cells were incubated with a rabbit polyclonal anti-FOXM1 (#5436, Cell Signaling) at a 1:100 dilution and a mouse monoclonal anti-Cyclin D1 antibody (#MA1-10324, Thermo Fisher Scientific Inc.) at a 1:100 dilution, overnight at 4 °C (the cells were only incubated with FOXM1 antibody in the negative control plate). The PLUS and MINUS secondary PLA probes against rabbit and mouse IgG were added and incubated for 1 h followed by incubation with ligase for 30 min at 37 °C. Amplification was then applied for 120 min at 37 °C. The coverslips were mounted on plates with Duolink Mounting Medium with DAPI (DUO82040, Sigma-Aldrich). The cells were imaged on a Zeiss LSM780-UV (Carl Zeiss Inc, Oberkochen, Germany) using a Plan-Apochromat 40X/1.40 oil DIC M27 objective. The number of PLA dots (Bustos et al. 2017 (link)) was measured by Fiji.
Yu L., Liu J., Yan Y., Burwell A., Castro L., Shi M, & Dixon D. (2021). “Metalloestrogenic” effects of cadmium downstream of G protein-coupled estrogen receptor and mitogen-activated protein kinase pathways in human uterine fibroid cells. Archives of Toxicology, 95(6), 1995-2006.
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3

Immunoprecipitation and Western Blotting

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Total proteins were extracted using the IP-lysis buffer (20 mM Tris-HCl, pH 8.0, 2mM EDTA, 137 mM NaCl, 1% NP-40) in the presence of 1X Halt protease inhibitor (Thermo Fisher) and 1 mM PMSF (Sigma, St Louis, MA). In total, 0.5–0.8 mg of protein from the lysates were incubated with 5 µg of bait antibodies, anti-P27KIP1 (Cell Signaling; 3686S) and anti-Cyclin D1 antibody (Invitrogen: MA5–12707) overnight at 4°C. Mouse (Cell Signaling, 5415S) or rabbit (Cell Signaling, 3900S) IgG1 isotype control was used. Protein immunocomplexes were then incubated with Protein G-agarose or protein A-agarose (Thermo Fisher) at 4°C up to 4H and were then washed 3 times with IP wash buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0,5% NP-40). Complex bound to the protein beads were eluted using 2X SDS buffer and were subjected to western blotting.
Knudsen E.S., Kumarasamy V., Nambiar R., Pearson J.D., Vail P., Rosenheck H., Wang J., Eng K., Bremner R., Schramek D., Rubin S.M., Welm A.L, & Witkiewicz A.K. (2022). CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports, 38(9), 110448.
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4

Kinase Assays for CDK2 and Cyclin D1

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CDK2 and cyclin D1 associated kinase reactions were performed as described in our previous study (Functional determinant). Protein extracts were prepared using kinase lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10 mM DTT, 10% glycerol) in the presence of protease inhibitors. Anti-cyclin D1 antibody (Invitrogen: MA5–12707) was used to immunoprecipitate cyclin D1 and its associated CDKs. The kinase reactions were performed using the specific buffer containing 50 mM HEPES-KOH, pH 7.5, 20 mM MgCl2, 1mM DTT in the presence of 2mM ATP. Recombinant RB C-terminal (10µg) was used as a substrate (Knudsen ES, Differential reglation).
To determine CDK2 kinase activity, cells were lysed using CDK2 kinase lysis buffer (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween-20). CDK2 complexes were immunoprecipitated using anti-CDK2 antibody (Santacruz; SC-6248). Kinase reactions were carried out in the reaction buffer ( 40 mM Tris-HCl pH 8, 20 mM MgCl2, 0.1 mg/mL BSA, 50 µM DTT).
Knudsen E.S., Kumarasamy V., Nambiar R., Pearson J.D., Vail P., Rosenheck H., Wang J., Eng K., Bremner R., Schramek D., Rubin S.M., Welm A.L, & Witkiewicz A.K. (2022). CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports, 38(9), 110448.
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5

Immunoprecipitation and Western Blotting

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Total proteins were extracted using the IP-lysis buffer (20 mM Tris-HCl, pH 8.0, 2mM EDTA, 137 mM NaCl, 1% NP-40) in the presence of 1X Halt protease inhibitor (Thermo Fisher) and 1 mM PMSF (Sigma, St Louis, MA). In total, 0.5–0.8 mg of protein from the lysates were incubated with 5 µg of bait antibodies, anti-P27KIP1 (Cell Signaling; 3686S) and anti-Cyclin D1 antibody (Invitrogen: MA5–12707) overnight at 4°C. Mouse (Cell Signaling, 5415S) or rabbit (Cell Signaling, 3900S) IgG1 isotype control was used. Protein immunocomplexes were then incubated with Protein G-agarose or protein A-agarose (Thermo Fisher) at 4°C up to 4H and were then washed 3 times with IP wash buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0,5% NP-40). Complex bound to the protein beads were eluted using 2X SDS buffer and were subjected to western blotting.
Knudsen E.S., Kumarasamy V., Nambiar R., Pearson J.D., Vail P., Rosenheck H., Wang J., Eng K., Bremner R., Schramek D., Rubin S.M., Welm A.L, & Witkiewicz A.K. (2022). CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports, 38(9), 110448.
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6

Kinase Assays for CDK2 and Cyclin D1

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CDK2 and cyclin D1 associated kinase reactions were performed as described in our previous study (Functional determinant). Protein extracts were prepared using kinase lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10 mM DTT, 10% glycerol) in the presence of protease inhibitors. Anti-cyclin D1 antibody (Invitrogen: MA5–12707) was used to immunoprecipitate cyclin D1 and its associated CDKs. The kinase reactions were performed using the specific buffer containing 50 mM HEPES-KOH, pH 7.5, 20 mM MgCl2, 1mM DTT in the presence of 2mM ATP. Recombinant RB C-terminal (10µg) was used as a substrate (Knudsen ES, Differential reglation).
To determine CDK2 kinase activity, cells were lysed using CDK2 kinase lysis buffer (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween-20). CDK2 complexes were immunoprecipitated using anti-CDK2 antibody (Santacruz; SC-6248). Kinase reactions were carried out in the reaction buffer ( 40 mM Tris-HCl pH 8, 20 mM MgCl2, 0.1 mg/mL BSA, 50 µM DTT).
Knudsen E.S., Kumarasamy V., Nambiar R., Pearson J.D., Vail P., Rosenheck H., Wang J., Eng K., Bremner R., Schramek D., Rubin S.M., Welm A.L, & Witkiewicz A.K. (2022). CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports, 38(9), 110448.
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