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Cyclin d1

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Cyclin D1 is a protein that plays a key role in the regulation of the cell cycle. It is an essential component of the cell division process and is involved in the transition from the G1 phase to the S phase of the cell cycle.

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Lab products found in correlation

β actin, by Merck Group (13 mentions) Gapdh, by Merck Group (6 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Tubulin, by Merck Group (5 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Cycloheximide (chx), by Merck Group (3 mentions) Caspase 3, by Cell Signaling Technology (3 mentions) Bcl 2, by Merck Group (3 mentions) Erk1 2, by Cell Signaling Technology (3 mentions) β actin, by Santa Cruz Biotechnology (3 mentions) Nitrocellulose membrane, by GE Healthcare (3 mentions) Eif4e, by Merck Group (2 mentions) Actin polyclonal antibodies, by Merck Group (2 mentions) P akt ser473, by Cell Signaling Technology (2 mentions) P egfr tyr1068, by Cell Signaling Technology (2 mentions) Erk1 2, by Merck Group (2 mentions) Tf receptor, by Abcam (2 mentions) Nonidet p 40, by Merck Group (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)

44 protocols using cyclin d1

1

Antibody Panel for Immunoblot Analysis

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Antibodies used for immunoblots include Rheb (Cell signalling, Cat# 1387, 1:1000), Cre (Cell signalling, Cat# 12830, IHC/IHF - 1:50), Cleaved Caspase 3 (Cell signaling, Cat# 9661, IHC - 1:500), pAkt S473 (Cell signaling, Cat# 4060, 1:1000), pAkt Thr308 (Cell signaling, Cat# 13038, 1:1000), pS6 S240/244 (Cell signaling, Cat# 5364, WB - 1:1000, IHC/IHF – 1:500), Total S6 (Cell signaling, Cat# 2317), p4EBP1 S65 (Cell signaling, Cat# 13443, WB-1:1000, IHC/IHF-1:500), p4EBP1 Thr37/46 (Cell signaling, Cat# 2855, WB-1:1000, IHC/IHF-1:500), 4EBP1 (Cell signaling, Cat# 9644, WB-1:1000), eIF4E (Cell signaling, Cat# 9742, 1:1000), Cyclin D1 (Cell signaling, Cat# 2978, 1:1000), Cyclin D1 (Cell signaling, Cat# 2922, IP-1:50), Cyclin D3 (Cell signaling, Cat# 2936, 1:1000), Actin (Sigma, A5316, 1:5000), PyV mT (clone 762, a gift from Dr. Steven Dilworth, IHC/IHF-1:100), Akt1/2 (Santa Cruz, SC-1619, 1:200), E-Cadherin (BD Bioscience, Cat# 610182, 1:1000), Neu (Dako, A0485, 1:500), Ki67 (Abcam, ab15580, 1:500), Cytokeratin 8/18 (Fitzgerald, 20R-CP004, 1:500), Cytokeratin 14 (BioLegend, PRB-155P, 1:500), and Anti-puromycin (Millipore, Clone 12010 – MABE343, 1:1000). Goat anti-rabbit IRDye 800, Goat anti-mouse IRDye 680, Donkey anti-goat IRDye 800 (Li-COR, 925–32211, 925–68070, 925–68074, 1:10 000)
Xiao B., Zuo D., Hirukawa A., Cardiff R.D., Lamb R., Sonenberg N, & Muller W.J. (2020). Rheb1-independent Activation of mTORC1 in Mammary Tumors Occurs Through Activating Mutations in mTOR. Cell reports, 31(4), 107571.
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2

Cellular Fractionation and Immunoblot Analysis

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Cells were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany). Nuclear and cytoplasmic fractions were performed using the ProteoJET Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany). Immunoblots were performed as described previously [19 (link)]. Supernatant was harvested 24h and 48h after treatment. Immunoblots were probed with primary antibodies against SSTR1-5 (abcam, Cambridge, UK), PARP, AKT, pAKT, ERK, pERK, pRPS6, RPS6, eiF4E, PCNA, Cyclin D1, CDK4, chromogranin A and beta-actin (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies against mouse or rabbit were obtained from Amersham (Freiburg, Germany) and goat from Sigma. Blots were detected by Western Lightning ECL (PerkinElmer, Rodgau, Germany).
Krug S., Mordhorst J.P., Moser F., Theuerkorn K., Ruffert C., Egidi M., Rinke A., Gress T.M, & Michl P. (2019). Interaction between somatostatin analogues and targeted therapies in neuroendocrine tumor cells. PLoS ONE, 14(6), e0218953.
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3

Western Blotting and Immunohistochemical Analysis

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Western blotting and immunohistochemical (fluorescence) staining were performed as previously described [37 (link), 38 (link)]. The primary antibodies used in this study were AHR (RPT9), HDAC8 (GeneTex), RB1, p53, Cyclin D1, PCNA, ARNT1, CYP1B1 and E2F1 (1:200), actin polyclonal antibodies (1:5000 dilution; Sigma–Aldrich), HA and GFP monoclonal antibodies (1:200 dilution; Upstate, NY, USA), FITC-conjugated anti-rabbit IgG, rhodamine-conjugated anti-mouse IgG, alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories), and Ki67 goat polyclonal antibody (1:200 dilution; Santa Cruz Biotechnology) [28 (link)]. All of the experiments were repeated at least three times, and staining with relevant control antibodies was performed in parallel and in all cases no signals were seen (for example, Supplementary Figure S1).
Wang L.T., Chiou S.S., Chai C.Y., Hsi E., Wang S.N., Huang S.K, & Hsu S.H. (2016). Aryl hydrocarbon receptor regulates histone deacetylase 8 expression to repress tumor suppressive activity in hepatocellular carcinoma. Oncotarget, 8(5), 7489-7501.
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4

Western Blotting for Protein Analysis

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The cells were harvested 24 h after treatment at the indicated doses and times, and the cell lysates were subjected to western blotting, performed as described previously (20 (link)). Briefly, the cells were collected and lysed using 10 mM Tris, 1 mM ethylenediaminetetraacetic acid, 10 mM KCl and 0.3% Triton X-100 (pH 7.9). The concentration of the protein samples was measured by the Bradford method. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Hybond-P polyvinylidene fluoride membranes (Amersham, Piscataway, NJ, USA). The membranes were subjected to western blot analysis with primary antibodies to caspase-8, -9 and -3, poly(ADP-ribose) polymerase (PARP), p110δ, Akt, phospho-Akt, p65, phospho-p65, cyclin-dependent kinase (CDK)4, CDK6, cyclin D1 and β-actin (Sigma-Aldrich). The secondary antibodies used in this study were provided by Multisciences Co., Ltd. (Hangzhou, China).
LIN S., LI J., ZHOU W., QIAN W., WANG B, & CHEN Z. (2014). BIIB021, an Hsp90 inhibitor, effectively kills a myelodysplastic syndrome cell line via the activation of caspases and inhibition of PI3K/Akt and NF-κB pathway proteins. Experimental and Therapeutic Medicine, 7(6), 1539-1544.
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5

Western Blot Analysis of Apoptosis Regulators

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Total protein of cells and tissue were extracted using a total protein extraction buffer (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat milk and incubated with the primary antibodies and then incubated with species-specific secondary antibodies. Image J software was used to quantify the results. Antibodies and their corresponding concentrations used were as follows: Krt17 (1:1000, Proteintech, USA), Bcl2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Bcl-xl (1:1000, CST, USA), Cleaved caspase-3 (1:1000, CST, USA), Cyclin D1 (1:1000, Sigma, USA), Cyclin E (1:1000, Sigma, USA), IRDye680 anti-mouse (1:20000, LI-COR) and IRDye800 anti-rabbit (1:10000, LI-COR). β-actin (1:1000, CST, USA) was used as a control to ensure the equal loading of protein.
Hu H., Xu D.H., Huang X.X., Zhu C.C., Xu J., Zhang Z.Z, & Zhao G. (2018). Keratin17 Promotes Tumor Growth and is Associated with Poor Prognosis in Gastric Cancer. Journal of Cancer, 9(2), 346-357.
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6

Multiprotein Expression Analysis in Cells

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Western blotting and immunohistochemical (fluorescence) staining were performed as described previously (Hsu et al. 2013 (link); Chiou et al. 2014 (link)). The primary antibodies used in this study were Cyclin D1 (1:200), and actin polyclonal antibodies (1:5000 dilution; Sigma-Aldrich). The AHR and Ki67 goat polyclonal antibody (1:200 dilution; Santa Cruz Biotechnology). The Ikaros1, E2F1, IDO1, ETV6, TDO2, IDO2, CYP1B1, AML1, OCT4, and Ikaros1 (1:500 dilution) primary antibody was obtained from GeneTex International Corp. All of the experiments were repeated at least 3 times.
Wang L.T., Liu K.Y., Wang S.N., Lin M.H., Liao Y.M., Lin P.C., Huang S.K., Hsu S.H, & Chiou S.S. (2022). Aryl hydrocarbon receptor–kynurenine axis promotes oncogenic activity in BCP-ALL. Cell Biology and Toxicology, 39(4), 1471-1487.
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7

Mammary Carcinoma Cell Line Stimulation and Protein Analysis

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MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF-7, BT-474 and SK-BR-3 mammary carcinoma cell lines were serum starved in DMEM containing 0.1% bovine serum albumin (BSA, SIGMA) and then stimulated with 10% FBS or 5% WF for the indicated time points.
The preparation of protein lysates and immunoblotting analysis was performed as previously described [33 (link)], except that membranes were blocked with Odyssey Blocking Buffer (Licor, Biosciences) and, following incubation with primary antibodies overnight at 4°C, incubated 1 hour at RT with IR-conjugated (Alexa Fluor 680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, Licor).
Primary antibodies directed against AKT (sc-1618), ERK1 (sc-94), STAT3 (sc-482), Fibrillarin (sc-25397) and Vinculin (sc-7694) were purchased from Santa Cruz Biotechnology, Inc.; pT202/204 ERK1/2 (#9101), pS473 AKT (#4060), pY705STAT3 (#9131) were purchased from Cell Signaling; Tubulin (#T9026) was purchased from SIGMA; Cyclin D1 (#04-1151) and Bcl2 (#OP60) were purchased from Millipore; Grb2 was purchased from BD Transduction Laboratories (#610112).
Segatto I., Berton S., Sonego M., Massarut S., Perin T., Piccoli E., Colombatti A., Vecchione A., Baldassarre G, & Belletti B. (2014). Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling. Oncotarget, 5(15), 6267-6279.
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8

Western Blot Analysis of Cellular Proteins

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Total protein was isolated from NP cells or samples with RIPA buffer (Sigma, MO, USA). A total of 20 μg of protein was separated with 10% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide) and transferred to membranes (Millipore, MA, USA). The membrane was incubated with the primary antibodies (SMAD3, catalogue: SAB4504210 and GAPDH, catalogue: G9545, Sigma; PCNA, catalogue: SAB2502098 and cyclin D1, catalogue: SAB1306586) at 4 °C overnight. Then, the membrane was labeled with horseradish peroxidase (HRP)-conjugated anti-IgG antibody. The protein signal was measured with the ECL system. GAPDH was used as the internal control.
Zhao K., Zhang Y., Yuan H., Zhao M, & Zhao D. (2019). Long noncoding RNA LINC00958 accelerates the proliferation and matrix degradation of the nucleus pulposus by regulating miR-203/SMAD3. Aging (Albany NY), 11(23), 10814-10825.
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9

Quantifying EGFR Signaling Dynamics

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Cells were lysed with Nonidet P-40 (1%, Sigma) that included protease inhibitor cocktail (Roche) and then centrifuged for 15 min at 14,000 r.p.m. at 4°C. Cell protein lysates (50-100 μg) were dissolved in loading buffer (62.5 mM Tris-HCl pH 6.8, 10% Glycerol, 2% SDS, 0.01% Bromophenol Blue, 100mM DTT), and separated by SDS-polyacrylamide gel electrophoresis. Antibodies used for western blotting were: Tf Receptor (Abcam ab38171, 1:250); EGFR (Millipore 06-847, 1: 250); p-EGFR, Tyr1068 (Cell Signaling 2234, 1:1000); ERK1/2 (Sigma M 5670, 1:2000); pERK1/2, Thr183 (Sigma M 7802, 1:500); Cyclin D1 (Sigma C 7464, 1:200); AKT (Cell Signaling 9272, 1:1000); and pAKT, Ser 473 (Cell Signaling 4056, 1:1000). Loading controls used were Tubulin (Sigma T 9026, 1:1000) and GAPDH (Sigma G 9295, 1:20,000).
Cells used for degradation assays were plated at 2.5 × 105 cells/well in a 6-well tissue culture dish and allowed to adhere overnight. The following day cells were growth factor starved for 2 hrs in the presence of 50 μM cycloheximide (Sigma). These conditions were maintained for the duration of the experiment. Following, 20 ng mL-1 EGF (PeproTech) was added to the media for the indicated amounts of time. Cells were then chilled on ice, washed with PBS, and protein lysates were collected for immunoblotting. Blots were cropped to show relevant bands.
Kondapalli K.C., Llongueras J.P., Capilla-González V., Prasad H., Hack A., Smith C., Guerrero-Cázares H., Quiñones-Hinojosa A, & Rao R. (2015). A Leak Pathway for Luminal Protons in Endosomes Drives Oncogenic Signaling in Glioblastoma. Nature communications, 6, 6289.
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10

Protein Expression in Human Pulmonary Artery

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Proteins were extracted from human pulmonary artery tissues or PAECs and lysed in RIPA lysis buffer (RIPA, Beyotime, China) in accordance with the instructions of the manufacturer. Protein concentrations were evaluated using the BCA assay based on the albumin standard. The primary antibodies were used as follows: Dec1 and PPARγ (Abcam, United States, dilution: 1/1,000, for both), Cyclin B1 and Cyclin D1 (Sigma, United States, dilution 1/1,000, for both), Bax (Cell Signaling Technology, United States, dilution: 1/1,000), Bcl-2 (Sigma, United States, dilution 1/1,000), cleaved caspase 3, and β-actin (Santa Cruz, United States, dilution 1/1,000, for both). Detailed information of Western blot was described previously (Li et al., 2020 (link)). β-actin was used as a loading control. Fold change of protein expression was evaluated as average fold change for the ratio of targeting genes/β-actin in treated samples compared with that in the controls. Measurements were repeated three times.
Li X., Liu C., Qi W., Meng Q., Zhao H., Teng Z., Xu R., Wu X., Zhu F., Qin Y., Zhao M., Xu F, & Xia M. (2021). Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension. Frontiers in Cell and Developmental Biology, 9, 757168.
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