Ccnd1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

CCND1 is a protein-coding gene that plays a role in cell cycle regulation. It encodes the cyclin D1 protein, which is involved in the progression of cells through the G1 phase of the cell cycle. CCND1 is a commonly used biomarker in various research applications.

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Anti-CCND1 (9 citations) Mouse anti-CCND1 (3 citations)

16 protocols using ccnd1

Western Blot Analysis of Cell Cycle Regulators

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One hundred microgram protein lysate per lane was used for Western blot analysis. Antibodies used in this study: RB1 (CusaBio PA003948), RB-P (Cell Signaling 9307), E2F1 (Santa Cruz SC-193), CDK2 (CusaBio PA001533), CDK4 (Santa Cruz SC-23896), CDK6 (Santa Cruz SC-53638), CCND1 (Santa Cruz SC-8396), CCNE2 (Proteintech 11,935–1-AP), CDKN2A (Prosci 4211), CDKN1A (Santa Cruz SC-6246), PCNA (Proteintech 10,205–2-AP) and GAPDH (Santa Cruz FL-335).

Hsu C.L., Lui K.W., Chi L.M., Kuo Y.C., Chao Y.K., Yeh C.N., Lee L.Y., Huang Y., Lin T.L., Huang M.Y., Lai Y.R., Yeh Y.M., Fan H.C., Lin A.C., Lu Y.J., Hsieh C.H., Chang K.P., Tsang N.M., Wang H.M., Chang A.Y., Chang Y.S, & Li H.P. (2018). Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 37, 233.

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Western Blotting Analysis of Cell Signaling Proteins

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Western blotting was performed using a SDS-PAGE Electrophoresis System according to the previous^{39 (link), 40 (link)} description with rabbit polyclonal anti-FAP antibody (1 : 1000; LifeSpan BioSciences Inc., Seattle, WA, USA), anti-β-actin, CCND1, CDK4, p21, E2F1, and p27 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-C-Myc, PTEN, β-catenin, GSK-3b, p-GSK-3b(ser-9), p-Erk1/2(Tyr202/Tyr204),Erk1/2, p-MEK1/2 (Ser-217/221), MEK1,CCNE1, MMP2, MMP9, p-RB (ser780), RB, AKT, p-Akt (Ser-473), PI3K, p-PI3K (Tyr458), p16, p27, Slug, Snail, Vimentin, N-cadherin, and E-cadherin (1 : 1000; Cell Signaling Technology, Danvers, MA, USA). Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).

Wang H., Wu Q., Liu Z., Luo X., Fan Y., Liu Y., Zhang Y., Hua S., Fu Q., Zhao M., Chen Y., Fang W, & Lv X. (2014). Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma. Cell Death & Disease, 5(4), e1155-.

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Immunohistochemical Detection of CTGF and CCND1

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Histologic sections were deparaffinized with xylene and ethanol. Antigen retrieval was performed using 10 mM citrate buffer, pH 6.0. The sections were blocked with 10% normal goat serum for 30 min, followed by an overnight incubation at 4°C with rabbit polyclonal antibody against CTGF (1:200) (Santa Cruz, CA, USA), or CCND1 (1:200) (Santa Cruz, CA, USA). A negative control, replacing the primary antibody with goat IgG at the same concentration, was included. Biotinylated goat anti-rabbit (diluted 1:200) (Santa Cruz, CA, USA) was followed by an incubation with streptavidin-peroxidase conjugate.

Wang R., Ding X., Zhou S., Li M., Sun L., Xu X, & Fei G. (2016). MicroRNA-26b attenuates monocrotaline-induced pulmonary vascular remodeling via targeting connective tissue growth factor (CTGF) and cyclin D1 (CCND1). Oncotarget, 7(45), 72746-72757.

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Protein Expression Profiling in GBM and GSCs

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GSCs and GBM cell lines were transfected with either miR-3928 or scrambled control using RNAiMAX. Total cell lysate was extracted from each sample 48 h after transfection. The protein concentrations were quantified using the Bradford protein assay according to established protocols. Immunoblots were performed according to standard protocols. Antibodies for the following proteins were used: p53 (sc-263; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), MDM2 (OP115; Cell Signaling Technology), CD44 (3570S, Cell Signaling Technology, Danvers, MA, USA), DDX3X (sc-81247, Santa Cruz Biotechnology), HMGA2 (8179S; Cell Signaling Technology), CCND1 (sc-8396, Santa Cruz Biotechnology), BRAF (sc-5284, Santa Cruz Biotechnology), ATOH8 (PA5-2710, Thermo Fisher Scientific), BMI1 (6964S, Cell Signaling Technology), p-p53(Ser15) (9286S, Cell Signaling Technology), and p-p53(Ser20) (9287S, Cell Signaling Technology). Every immunoblot was also immunostained with GAPDH (Santa Cruz Biotechnology) as a loading control. All antibodies were used at a dilution of 1:500.

Mulcahy E.Q., Zhang Y., Colόn R.R., Cain S.R., Gibert MK J.r., Dube C.J., Hafner M, & Abounader R. (2022). MicroRNA 3928 Suppresses Glioblastoma through Downregulation of Several Oncogenes and Upregulation of p53. International Journal of Molecular Sciences, 23(7), 3930.

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Protein Expression Analysis Protocol

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The cells were lysed in radioimmunoprecipitation lysis buffer (Beyotime), and the protein concentrations were determined. Approximately 60 μg of protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The primary antibodies used were as follows: YAP (sc-15407; Santa Cruz Biotechnology), Cyr61 (sc-13100; Santa Cruz Biotechnology), CTGF (sc-101586; Santa Cruz Biotechnology), CCND1 (sc-20044; Santa Cruz Biotechnology), LC3B and Beclin1 (3868S and 3738, respectively; Cell Signaling Technology), cleaved PARP and caspase 3 (9541 and 9661, respectively; Cell Signaling Technology), and Atg-3 and -5 (3415 and 2630, respectively; Cell Signaling Technology).

Xiao L., Shi X.Y., Zhang Y., Zhu Y., Zhu L., Tian W., Zhu B.K, & Wei Z.L. (2016). YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells. OncoTargets and therapy, 9, 1105-1114.

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Western Blot Analysis of Protein Lysates

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Protein lysates (40 μg) were prepared in the presence of protease inhibitors and analyzed as described previously (18 (link)) using 4–20% Criterion gels (Bio-Rad, Hercules, CA, USA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (YAP1, RB1, CDKN2A/B, NEUROD1, CCND1, CDK4, CDK6, pRB-s780), Santa Cruz (Dallas, TX, USA) (INSM1, CDK2, E2F1), Sigma (St. Louis, MO, USA) (Actin, POU2F3), and BD Biosciences (Franklin Lakes, NJ, USA) (ASCL) and are listed in Sup. Table 1. Bands on western blots were quantified using Image J.

Wildey G., Shay A.M., McColl K.S., Yoon S., Shatat M.A., Perwez A., Spainhower K.B., Kresak A.M., Lipka M., Yang M., Behtaj M., Fu P., Alahmadi A., Mneimneh W., Abbas A, & Dowlati A. (2023). Retinoblastoma expression and targeting by CDK4/6 inhibitors in small cell lung cancer. Molecular cancer therapeutics, 22(2), 264-273.

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Silencing Cell Cycle Regulators in Pancreatic Cells

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Pancreatic cell lines were seeded in a 6-well plate at a desity of 5×10⁵ cells per well and transfected with siRNA against either CCND1 (Santa Cruz biotechnology), CCNE1 (SMARTpool: Accell CCNE1 siRNA; Dharmacon) and PLK1(SMARTpool: Accell PLK1 siRNA; Dharmacon) at a final concentration 50nmol/L with Lipofectamine RNAiMAX(Invitrogen) according to manufacture's instructions. Transfections with a control sRNA(TMEM) served as a negative control.

Franco J., Witkiewicz A.K, & Knudsen E.S. (2014). CDK4/6 inhibitors have potent activity in combination with pathway selective therapeutic agents in models of pancreatic cancer. Oncotarget, 5(15), 6512-6525.

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Subcellular Protein Analysis Workflow

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Cells were grown to 70–80% confluency and were treated or transduced as indicated in the figures. Thereafter, cells were lysed using 1× RIPA (Cell Signaling Technologies, 9806) with protease inhibitors (coMplete, Roche, 42484600) and phosphatase inhibitors (PhosSTOP, Roche, 04906837001). For experiments using nuclear and cytoplasmic fractions, fractions were extracted using NE-PER (ThermoFisher Scientific, 78835). Using 10% or 4–12% SDS-PAGE gels, gels were transferred onto PVDF (Millipore, IPFL00010) or nitrocellulose membranes (ThermoFisher Scientific, IB23001). Blots were then visualized using Odyssey Classic v3.0.30 (Licor, Lincoln, NE). Antibodies used in this study include: XPO1 (Santa Cruz; sc-5595), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), p53 (Santa Cruz; sc-126), TRIP13 (Abcam; ab128171), α-Tubulin (Santa Cruz, sc-5286), α-Tubulin (Cell Signaling; 2144), Lamin A/C (Cell Signaling; 4777 or 2032), p21 (Cell Signaling; 29475), CCND1 (Santa Cruz; sc-8396). Results shown are representative of at least two biological replicates.

Mittal K., Cooper G.W., Lee B.P., Su Y., Skinner K.T., Shim J., Jonus H.C., Kim W.J., Doshi M., Almanza D., Kynnap B.D., Christie A.L., Yang X., Cowley G.S., Leeper B.A., Morton C.L., Dwivedi B., Lawrence T., Rupji M., Keskula P., Meyer S., Clinton C.M., Bhasin M., Crompton B.D., Tseng Y.Y., Boehm J.S., Ligon K.L., Root D.E., Murphy A.J., Weinstock D.M., Gokhale P.C., Spangle J.M., Rivera M.N., Mullen E.A., Stegmaier K., Goldsmith K.C., Hahn W.C, & Hong A.L. (2024). Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin. Communications Biology, 7, 426.

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Antibody and Chemical Analysis of Cell Cycle Regulation

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The following antibodies were used: pRB-S807/811 (Cell Signaling Technology, Danvers, MA), pRBL2-S672, RBL2 (Abgent, Inc., San Diego, CA), CCND1, CDK4, CDK6, GAPDH, β-ACTIN (Santa Cruz, Dallas, TX), RB (BD Biosciences, San Jose, CA), and γH2AX (Millipore, Billerica, MA). The following chemicals were used: PD-0332991 (Selleck Chemicals, Houston, TX), hydroxyurea, cytarabine, and aphidicolin (Sigma, St. Louis, MO).

Mohanty S., Mohanty A., Sandoval N., Tran T., Bedell V., Wu J., Scuto A., Murata-Collins J., Weisenburger D.D, & Ngo V.N. (2016). Cyclin D1 Depletion Induces DNA Damage in Mantle Cell Lymphoma Lines. Leukemia & lymphoma, 58(3), 676-688.

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Comprehensive Protein Expression Analysis

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Western blotting was performed with a SDS-PAGE Electrophoresis System according to previous reports [7 (link),13 (link)] with the following antibodies: rabbit polyclonal CCDC19 (Proteintech Group, Inc. Chicago, IL, USA), ACTB, GAPDH, CCND1, p21, E2F1, p15 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), C-Myc, C-Jun, AKT, p-Akt (Ser-473), PI3K, p16, p27, E-Cadherin and pPI3K (Tyr458; 1:1000; Cell Signaling Technology, Danvers, MA, USA). Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).

Liu Z., Mai C., Yang H., Zhen Y., Yu X., Hua S., Wu Q., Jiang Q., Zhang Y., Song X, & Fang W. (2014). Candidate tumour suppressor CCDC19 regulates miR-184 direct targeting of C-Myc thereby suppressing cell growth in non-small cell lung cancers. Journal of Cellular and Molecular Medicine, 18(8), 1667-1679.

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