Cdk4 cyclin d1

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CDK4/Cyclin D1 is a protein complex composed of two subunits: Cyclin-dependent kinase 4 (CDK4) and Cyclin D1. This complex plays a crucial role in the regulation of the cell cycle, specifically during the G1 phase. It functions as a serine/threonine protein kinase, catalyzing the phosphorylation of various target proteins involved in cell cycle progression.

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3 protocols using cdk4 cyclin d1

Phosphorylation of NFATc3 by CDK4/6

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NMR experiments were performed on a Varian (Agilent DD2 700) spectrometer equipped with a cryogenically cooled probe, and the spectrum was recorded at 287 K. CDK4/cyclin D1 and CDK6/cyclin D3 kinases were purchased from Signalchem. The phosphorylation reaction was performed with a sample containing 0.1mM ¹⁵N labeled NFATc3 with the addition of 10 µg CDK4 or 10 µg CDK6 in kinase reaction buffer [50 mM MES (pH 6.7), 140 mM NaCl, 10Mm MgCl₂, 0.1Mm EDTA, 2mM ATP and 5 mM DTT]. First, a control experiment of unphosphorylated NFATc3 in same kinase reaction buffer was recorded followed by addition of kinases and phosphorylation was monitored by using 2D ¹⁵N-HSQC experiments. In the inhibition assay, ~ 0.7µM of CKD6 was pre-incubated with 7µM inhibitor before addition to ¹⁵N labeled NFATc3 sample. Here, 2.5 ul of a 1mM stock of the inhibitor was added to 350 ul of the NMR sample. In a control experiment, the same amount of DMSO (2.5µL) was added. All spectra were processed using nmrPipe and analyzed with CcpNmr-Analysis (version 2.4.1).

Deng J., Wang E.S., Jenkins R.W., Li S., Dries R., Yates K., Chhabra S., Huang W., Liu H., Aref A.R., Ivanova E., Paweletz C.P., Bowden M., Zhou C.W., Herter-Sprie G.S., Sorrentino J.A., Bisi J.E., Lizotte P.H., Merlino A.A., Quinn M.M., Bufe L.E., Yang A., Zhang Y., Zhang H., Gao P., Chen T., Cavanaugh M.E., Rode A.J., Haines E., Roberts P.J., Strum J.C., Richards W.G., Lorch J.H., Parangi S., Gunda V., Boland G.M., Bueno R., Palakurthi S., Freeman G.J., Ritz J., Haining W.N., Sharpless N.E., Arthanari H., Shapiro G.I., Barbie D.A., Gray N.S, & Wong K.K. (2017). CDK4/6 Inhibition Augments Anti-Tumor Immunity by Enhancing T Cell Activation. Cancer discovery, 8(2), 216-233.

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Colorectal and Breast Cancer Cell Line Cultivation and Kinase Assay

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HCT-116 (colorectal carcinoma), HT-29 (colorectal adenocarcinoma) and MDA-MB-231 (breast adenocarcinoma) cell lines were purchased from American Type Culture Collection (ATCC). MDA-MB-231 cells were grown in RPMI media while HCT-116 and HT-29 cells were cultured in McCoy's 5A media, both supplemented with 10% FBS and antibiotics for 24 h before treatment with specified compounds for indicated times. Authentication of cell lines was done by ATCC through their DNA-STR profile. CDK1/Cyclin B1, CDK2/Cyclin A2, CDK4/Cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem. Aspirin, salicylic acid and trypsin-EDTA solution were purchased from Sigma-Aldrich; H1 Histones were obtained from EMD Millipore; [³²P] γ-ATP from PerkinElmer; 2,3-DHBA, 2,5-DHBA and all other reagents mentioned in this study were obtained from Thermo Fischer Scientific, Inc.

Sankaranarayanan R., Valiveti C.K., Dachineni R., Kumar D.R., Lick T, & Bhat G.J. (2019). Aspirin metabolites 2,3-DHBA and 2,5-DHBA inhibit cancer cell growth: Implications in colorectal cancer prevention. Molecular Medicine Reports, 21(1), 20-34.

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Protein Expression and Kinase Assay

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anti-p21^Cip1, anti-p27^Kip1, anti-CDK1, anti-CDK2, anti-CDK4, anti-cyclinA2, anti-cyclinB1, anti-cyclinD1, and anti-β tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-SLC5A8 from Invitrogen (Carlsbad, CA, USA); goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad (Hercules, CA, USA). CDK1/Cyclin B1, CDK2/Cyclin A2, CDK4/Cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).

Sankaranarayanan R., Valiveti C.K., Kumar D.R., Van slambrouck S., Kesharwani S.S., Seefeldt T., Scaria J., Tummala H, & Bhat G.J. (2019). The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention. Cancers, 11(3), 427.

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