Rabbit anti cyclin d1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Rabbit anti-cyclin D1 is a primary antibody that specifically binds to the cyclin D1 protein. Cyclin D1 is a key regulator of the cell cycle and plays a crucial role in the G1/S phase transition. This antibody can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the expression and localization of cyclin D1 in biological samples.

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13 protocols using rabbit anti cyclin d1

Western Blot Analysis of Protein Expression

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Samples containing equal amounts of protein were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels, subjected to electrophoresis and subsequently blotted onto nitrocellulose membrane (Millipore, Bedford, MA, USA). Membranes were blocked with tris-buffered saline buffer, pH 7.4, containing 0.1% Tween 20 and 5% skim milk and then incubated overnight at 4 °C with various primary antibodies in tris-buffered saline containing 0.1% Tween 20. The antibodies included mouse anti-p21 (1:2000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-β-actin (1:2000 dilution; Sigma–Aldrich), rabbit anti-cyclin D1 (1:2000 dilution, Santa Cruz Biotechnology), rabbit anti-phosphorylated p44/42MAPK (1:1000 dilution; Cell Signaling Technology), rabbit anti-p27 (1:1000 dilution; Cell Signaling), rabbit anti-β-catenin (1:1000 dilution; Cell Signaling) and rabbit anti-MMP-9 (1:1000 dilution; Millipore, Temecula, CA, USA). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution; Cell Signaling). The blots were detected with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and a bio-imaging analyzer (Fujifilm LAS-4000). The density of the respective bands was quantified by densitometric scanning of the blots using Image-Pro software (Media Cybermetrics, Inc., Bethesda, MD, USA).

Tsai Y.C., Lee Y.M., Hsu C.H., Leu S.Y., Chiang H.Y., Yen M.H, & Cheng P.Y. (2015). The effect of ferulic acid ethyl ester on leptin-induced proliferation and migration of aortic smooth muscle cells. Experimental & Molecular Medicine, 47(8), e180-.

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Western Blotting Quantification Procedure

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Samples were run out in 10% SDS‐PAGE, subsequently transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA) and blocked in Tris‐buffered saline (10 mmol/l Tris‐HCl, 150 mmol/l NaCl, pH 8.00) with 0.05% Tween 20 (TBS‐T) containing 5% non‐fat dry milk for 1 hr at room temperature. Blots were then incubated overnight at 4°C with rabbit anti‐phosphorylated p44/42MAPK (1:1000 dilution; Cell Signaling Technology), rabbit anti‐cyclin D1 (1:2000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti‐p21 (1:2000 dilution; Santa Cruz Biotechnology), anti‐MMP‐2 (1:1000 dilution; Millipore, Temecula, CA, USA) and mouse anti‐β‐actin (1:2000 dilution; Sigma‐Aldrich) antibodies. The membranes were incubated with HRP‐conjugated secondary antibodies (1:1000 dilution; Cell Signaling Technology). The blots were detected with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and a bio‐imaging analyser (Fujifilm LAS‐4000; GE Healthcare Life Sciences, Marlborough, MA, USA). Densitometric analysis was conducted with Image‐Pro software (Media Cybermetrics, Inc., Bethesda, MD, USA).

Tsai Y., Leu S., Peng Y., Lee Y., Hsu C., Chou S., Yen M, & Cheng P. (2016). Genistein suppresses leptin‐induced proliferation and migration of vascular smooth muscle cells and neointima formation. Journal of Cellular and Molecular Medicine, 21(3), 422-431.

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Osteoblast and Chondrocyte Gene Analysis

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Osteoblast and chondrocyte genes were analyzed by immunofluorescence using the following primary antibodies: rabbit-anti-Ihh (Abcam, ab39634), rabbit-anti-Cyclin D1 (Santa Cruz, sc-753), mouse-anti-PTHrP-R (Santa Cruz, sc-12722); and these secondary antibodies: FITC-goat-anti-mouse IgG(H+L) and TR-goat-anti-rabbit IgG (H+L). Imaging was taken by Leica Confocal Microscope and Zeiss fluorescent microscope.

Wu M., Li Y.P., Zhu G., Lu Y., Wang Y., Jules J., McConnell M., Serra R., Shao J.Z, & Chen W. (2014). Chondrocyte-specific Knockout of Cbfβ Reveals the Indispensable Function of Cbfβ in Chondrocyte Maturation, Growth Plate Development and Trabecular Bone Formation in Mice. International Journal of Biological Sciences, 10(8), 861-872.

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Antibody Reagents for Protein Analysis

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Affinity-purified rabbit anti-11βHSD2 (catalog no. BHSD22-A) was purchased from Alpha Diagnostic International; rabbit anti-murine COX-2 (catalog no. 160106) was from Cayman Chemicals; rabbit anti-CHOP (catalog no. 2895), rabbit anti-cleaved caspase-3 (catalog no. 9661), rabbit anti-p-ERK (T202/Tyr204, catalog no. 4370), p-mTOR (Ser2448, catalog no 2976), p-PERK (Thr980, Catalog no 3179) and p-eIF2α (Ser51, catalog no 9721) were from Cell Signaling; rabbit anti-cyclin D1 (catalog no. SC-753) was from Santa Cruz Biotechnology; mouse anti-mannose receptor (MR, CD206, catalog no.MAB25341) was from R&D; rabbit anti-Ki67 (ab15580) was from Abcam.

Chang J., Xue M., Yang S., Yao B., Zhang B., Chen X., Pozzi A, & Zhang M.Z. (2015). Inhibition of 11β-Hydroxysteroid Dehydrogenase Type II Suppresses Lung Carcinogenesis by Blocking Tumor COX-2 Expression as Well as the ERK and mTOR Signaling Pathways. PLoS ONE, 10(5), e0127030.

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Western Blot Immunodetection Assay

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RIPA buffer (KeyGEN Biotech.) was used to lyse the cells, and protein concentration was quantified by the BCA method. An equal amount of the extracted protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA for 1 h at room temperature and then incubated with the primary antibodies overnight at 4°C. After three washes with TBST, the membranes were incubated with secondary antibodies (1:3,000) for 1 h at room temperature. The membranes were washed with TBST three times before visualization by a chemiluminescence system. Antibodies used in this work: Mouse anti-CDK2 (SC-6248, Santa Cruz Biotechnology), rabbit anti-CDK4 (SC-260, Santa Cruz Biotechnology), rabbit anti-Cyclin D1 (SC-718, Santa Cruz Biotechnology), mouse anti-c-Myc (ab32, Abcam), rabbit anti-ERK (4695S, Cell Signaling Technology), rabbit anti-P-ERK (4370S, Cell Signaling Technology), rabbit anti-AKT (4685S, Cell Signaling Technology), rabbit anti-P-AKT (4060L, Cell Signaling Technology), rabbit anti-GAPDH (47724, Santa Cruz Biotechnology), rabbit anti-cleaved-Caspase-3 (9661S, Cell Signaling Technology), rabbit anti-Caspase-3 (9662S, Cell Signaling Technology), rabbit anti-cleaved-PARP1 (CY5035, Abways Technology), mouse anti-PARP1 (SC-74469X, Santa Cruz Biotechnology), HRP-Ms (7076, Cell Signaling Technology), and HRP-Rb (7074, Cell Signaling Technology).

Zhao X., Guo X., Yue W., Wang J., Yang J, & Chen J. (2017). Artemether suppresses cell proliferation and induces apoptosis in diffuse large B cell lymphoma cells. Experimental and Therapeutic Medicine, 14(5), 4083-4090.

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Western Blot Protocol for Protein Analysis

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Cells were washed twice with ice-cold PBS and lysed with buffer containing Tris-HCl (50 mM, pH = 7.4), NP-40 (1 %), Na-deoxycholate (0.25 %), NaCl (150 mM), EDTA (1 mM), PMSF (1 mM), Na₃VO₄ (1 mM), and NaF (1 mM) for total extract. Protein concentration was determined by the BCA protein assay (Pierce Chemical Co.). Equal amounts of cell lysates were separated by 10 % SDS-polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. Membranes were then incubated in blocking solution (5 % nonfat milk in 20 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween-20) (TBS-T), followed by incubation with the indicated antibodies at 4 °C overnight. The membranes were then washed in TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence (ECL) Western blotting substrate (Pierce) was used to detect the immunoreactive signals with an ECL-based FluorChem FC2 image system (Alpha Innotech). Rabbit anti-CIRBP was purchased from ProteinTech, and rabbit anti-Cyclin D1 was from Santa Cruz Biotechnology. All Western blotting analyses were performed in triplicates. FluorChem FC2 software was used to analyze the gray values of the bands in each group.

Zhang Q., Wang Y.Z., Zhang W., Chen X., Wang J., Chen J, & Luo W. (2016). Involvement of Cold Inducible RNA-Binding Protein in Severe Hypoxia-Induced Growth Arrest of Neural Stem Cells In Vitro. Molecular Neurobiology, 54(3), 2143-2153.

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Targeting Survivin and mTOR Signaling

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Sources were: YM155, BEZ-235 (Selleck Chemicals); rabbit anti-Survivin IgG (#AF886) (R&D Systems); rabbit anti-P-Rb (S807/811, #9308), mouse anti-Cyclin D1 (#2926), rabbit anti-Mcl-1(#5453), rabbit anti-P-p70S6K1 (T389, #9205), rabbit anti-Raptor (#4978, #4972) rabbit anti-P-Raptor (S792, #2083), rabbit anit-HIF-1α (#3716), rabbit anti-HIF-2α (#7096), rabbit anti-VHL (#2738), rabbit anti-P-AMPKα (T172, #2535), rabbit anti-Cyclin D1 (sc-753), rabbit anti-Cyclin D2 (sc-593), mouse anti-P-rS6 (S240, sc-293143), mouse anti-rS6 (sc-74459), mouse anti-GAPDH (sc-51907), mouse anti-AMPK-α1 (39881) (Santa Cruz Biotechnologies, Inc.); mouse anti-β-actin (#A-5441), dimethyoxalylglycine (400091), deferoxamine mesylate (D9533) (Sigma-Aldrich, Inc.); DMEM/F12 (Media Tech); characterized fetal bovine serum (FBS) (HyClone); Roscovitine (LC labs).

Danielpour D., Corum S., Welford S.M, & Shankar E. (2021). Hypoxia represses early responses of prostate and renal cancer cells to YM155 independent of HIF-1α and HIF-2α. Current Research in Pharmacology and Drug Discovery, 3, 100076.

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Protein Expression Analysis by Western Blot

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SDS-PAGE and electroblotting were carried out as usual. For Western blotting, the following antibodies were used: rabbit anti-BCL, Cell Signalling #2872; mouse anti c-Myc (9E10), SantaCruz #sc-040; rabbit anti-GAPDH, Cell Signalling #2118; rabbit anti-eIF4E-BP1, Cell Signalling #9644; rabbit anti-phospho-eIF4E-BP1, Cell Signalling #9459; rabbit anti-eIF4E, Cell Signalling #9742; rabbit anti-eIF2A (D7D2) XP, Cell Signalling #5324; mouse anti-eIF2A, Cell Signalling #2103; rabbit anti-cyclin D1, SantaCruz #753; mouse anti-β-actin, Sigma #A3854; (primary); anti-mouse-HRP conjugate, 1:5,000, Dako P0447; anti-rabbit-HRP conjugate, 1:10,000, GE Healthcare NA9340V.

Soukarieh F., Nowicki M.W., Bastide A., Pöyry T., Jones C., Dudek K., Patwardhan G., Meullenet F., Oldham N.J., Walkinshaw M.D., Willis A.E, & Fischer P.M. (2016). Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation. European Journal of Medicinal Chemistry, 124, 200-217.

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Western Blot Analysis of Protein Expression

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Protein extracts, separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, Clifton, NJ, USA), were probed with antibodies against KRT-14 (1:1000, ProteinTech, Chicago, IL). The following antibodies have been used: rabbit anti-cyclin D1 (0.4 μg/ml) and rabbit anti-GAPDH (0.2 μg /ml) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The antibodies anti-KRT14, and mouse anti-E2F1 (5 μg /ml) were purchased from BD Bioscience Pharmingen (San Jose, California, USA). After incubation, the corresponding anti-rabbit or anti-mouse secondary HRP-conjugated antibody was used (Santa Cruz Biotechnology) and the blots developed using an enhanced chemiluminescent substrate (Supersignal West Pico, Pierce, Rockford, IL).

Confalonieri M., Buratti E., Grassi G., Bussani R., Chilosi M., Farra R., Abrami M., Stuani C., Salton F., Ficial M., Confalonieri P., Zandonà L, & Romano M. (2017). Keratin14 mRNA expression in human pneumocytes during quiescence, repair and disease. PLoS ONE, 12(2), e0172130.

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Comprehensive Western Blot Analysis

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Western blot analysis was performed as described previously [41 (link)] using following antibodies: mouse anti-VRK1 (Santa Cruz, sc-271062), goat anti-actin (Santa Cruz, sc-1615), rabbit anti-cyclin D1 (Santa Cruz, sc-753), rabbit anti-cyclin D2 (Santa Cruz, sc-181), rabbit anti-CREB (Cell signaling, 9198), rabbit anti-p-CREB (Cell signaling, 9191), rabbit anti-p27 (Santa Cruz, sc-528), mouse anti-p21 (Santa Cruz, sc-6246), rabbit anti-p-Rb^S795(Cell signaling, 9301), mouse anti-p53 (Santa Cruz, sc-126) and rabbit anti-p-Rb^S807/S811(Cell signaling, 9308).

Lee N., Kwon J.H., Kim Y.B., Kim S.H., Park S.J., Xu W., Jung H.Y., Kim K.T., Wang H.J, & Choi K.Y. (2015). Vaccinia-related kinase 1 promotes hepatocellular carcinoma by controlling the levels of cell cycle regulators associated with G1/S transition. Oncotarget, 6(30), 30130-30148.

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