Cyclin b

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Cyclin B is a key regulatory protein involved in the cell cycle. It plays a crucial role in the transition from the G2 phase to the M phase of the cell cycle, driving the activation of the cyclin-dependent kinase (CDK) complex. Cyclin B binds to and activates CDK1, which in turn phosphorylates various substrates to initiate and coordinate mitotic events.

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Close spelling variants of this product

Cyclin B (sc-752; Anti-cyclin B Cyclin A and B Anti-cyclin B antibody

33 protocols using cyclin b

Western Blot Analysis of Cell Signaling

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Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

WOO S.M., CHOI Y.K., KIM A.J., CHO S.G, & KO S.G. (2015). p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells. Molecular Medicine Reports, 13(2), 1091-1096.

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Western Blotting and Immunofluorescence Protocols

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For Western blotting, whole cell lysates were prepared by lysing cell pellets directly in sodium dodecyl (SDS)-polyacrylamide gel electrophoresis buffer. For immunofluorescence with the Mcm3 antibody, HeLa cells were pre-extracted with phosphate-buffered saline (PBS) containing 0.5% (v/v) Triton X-100, and fixed in 4% paraformaldehyde solution (WAKO) for immunostaining. Images were acquired by Keyence BZ-8100 and nuclear staining intensity was measured by Dynamic Cell count software (Keyence). The following primary antibodies were used: Cdt1[6 (link)], Cdt2[50 (link)], Mcm2 (lab stock), Mcm3 (ab4460, Abcam, Cambridge, UK), Mcm4 (lab stock), Mcm6 (Santa Cruz Biotechnology, Dallas, TX), Cyclin A (mouse, Ab-6, Neomarkers; rabbit, H-432, Santa Cruz Biotechnology), Cyclin B (H433, Santa Cruz Biotechnology), PCNA (PC10, Santa Cruz Biotechnology), caspase-3 (96625, Santa Cruz Biotechnology), RCC1 (lab stock), Chk1 pS296 (Cell Signaling Technology, Danvers, MA), Chk2 pT68 (Cell Signaling Technology), Cdk2 pT160 (Cell Signaling Technology), and CPD (Cosmo Bio, Tokyo, Japan). Protein levels were analyzed by ImageJ software.

Morino M., Nukina K., Sakaguchi H., Maeda T., Takahara M., Shiomi Y, & Nishitani H. (2015). Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response. PLoS ONE, 10(3), e0120553.

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Comprehensive Immunofluorescence Analysis of Microtubule-Targeting Agents

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The following antibodies were used in this study: phospho-histone H3 (Ser10)-488 (p-H3-488, Cell Signaling), ɑ-tubulin (Serotec), Anti-Centromere-Antibodies (ACA, Cortex Biochem), Cyclin B (Santa Cruz), BubR1 (Abcam) and Bub1 (EMD Millipore). Affinipure FITC-, Cy3- and Cy5-conjugated secondary antibodies were from Jackson Immuno Research. The following reagents were used in this study: CellTiter-Glo Luminescent Cell Viability Assay (Promega), Caspase-Glo 3/7 (Promega), HTS-Tubulin polymerization assay kit (Cytoskeleton Inc), ProLong Gold anti-fade reagent (Invitrogen), Hoechst 33342 (Thermo Scientific) and Vybrant DyeCycle Green (Invitrogen). Microtubin-15-27 were purchased from MolPort at >95% purity by LCMS. Microtubin-1-14 were synthesized and purified to >95% purity.

Senese S., Lo Y.C., Gholkar A.A., Li C.M., Huang Y., Mottahedeh J., Kornblum H.I., Damoiseaux R, & Torres J.Z. (2017). Microtubins: a novel class of small synthetic microtubule targeting drugs that inhibit cancer cell proliferation. Oncotarget, 8(61), 104007-104021.

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Cell Line Characterization and Antibody Validation

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The HeLa cell line (BCRC-60005), C33A cell line (BCRC-60554), DLD-1 cell line (BCRC-60132), LS174T (BCRC-60053), HA22T (BCRC-60168), and HA59T (BCRC-60169) were purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. HeLa, C33A, and LS174T Cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100-U/mL streptomycin, and 100-mg/ml penicillin. The DLD-1 cells were maintained in Roswell Park Memorial Institute medium supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin. HA22T and HA59T were maintained in MEM supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin (all from Gibco). Primary antibodies targeting the following proteins were used: CDK1, CDK2, cyclin A, cyclin B, cyclin E, p53, and corresponding secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) (all from Santa Cruz); β-actin, ROGDI, p21, γ-H2A.X (phospho S140) and γ-H2A.X (phospho S139) (all from Abcam); and ROGDI (Proteintech, 17047-1-AP).

Chen Y.F., Cho J.J., Huang T.H., Tseng C.N., Huang E.Y, & Cho C.L. (2016). Downregulation of a novel human gene, ROGDI, increases radiosensitivity in cervical cancer cells. Cancer Biology & Therapy, 17(10), 1070-1078.

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Protein Expression and Analysis

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Cells were split by radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) add with protease inhibitor (Pierce, Rockford, IL, USA). After centrifugation removal cell debris, add 1/4 volume of lysis buffer to the lysates, and then boiling 10 min in water. The total protein sample was pointed into and separated in the SDS-polyacrylamide gel. Then transferred it into a PVDF membrane (Millipore, Boston, MA, USA). Next, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. Protein bands were exposure by chemiluminescence reagents (Millipore) and quantified using the Image Lab Image Document. Antibodies PPARγ, ATGL (Cell Signaling, Boston, MA, USA), KLF9 (Abcom, Cambridge, UK), aP2, FAS, cyclin B, cyclin D, cyclin E, p27, ZEB1 (Santa Cruz, Dallas, TX, USA), GAPDH (Boster, Wuhan, China), β-tublin (Sungene Biotech, Tianjin, China) were used.

Peng Y., Chen F.F., Ge J., Zhu J.Y., Shi X.E., Li X., Yu T.Y., Chu G.Y, & Yang G.S. (2016). miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes. International Journal of Molecular Sciences, 17(12), 2047.

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Comprehensive Cell Cycle Regulation Assay

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The monoclonal or polyclonal antibodies against cyclin A, cyclin B, cyclin C, cyclin D, cyclin E, CDK2, CDK4, CDK6, p16, p18 and p27 were purchased from Santa Cruz Biotechnology. The monoclonal antibodies against p21 and p57 were obtained from BD Biosciences. Horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. RNA and DNA extract kits, RNA RT and polymerase chain reaction (PCR) kits were obtained from Qiagen. NorthernMAX kit was from Ambion. Immunoblotting was performed using the Enhanced chemiluminescence (ECL) western blot detection kit (Amersham Biosciences). Biotin Chromogenic Detection kit was from Thermo Scientific. Protein A-Sepharose 4B Conjugate and Dynabeads MyOne Streptavidin C1 magnetic beads were obtained from Invitrogen. The cell lines used in this study were from American Type Culture Collection (ATCC).

Du W.W., Yang W., Liu E., Yang Z., Dhaliwal P, & Yang B.B. (2016). Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Research, 44(6), 2846-2858.

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Organoid Drug Treatment and Protein Analysis

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Organoids were passaged and cultured in 6-well plate for one day before adding drugs (5 μM gefitinib or 50 nM docetaxel or vehicle). For gefitinib inhibitor experiment, organoids were collected for total protein extraction at 12 and 24 h after treatment, and for docetaxel, at 24 h and 48 h. The protein bands were visualized by using Immobilon Western Chemiluminescent HRP Substrate (Millipore) under ChemiDoc Touch Imaging System (Bio-Rad). The antibodies used were listed as following: Phospho-AKT (1:1000, CST, 4060 S), AKT (1:1000, CST, 9272 S), Phospho-ERK (1:1000, CST, 4370 S), ERK (1:1000, CST, 4695 S), Phospho-STAT3 (1:1000, CST, 9145 S), STAT3 (1:1000, CST, 9139 S), Cyclin A (1:200, Santa Cruz, sc-751), Cyclin B (1:200, Santa Cruz, sc-752), Phospho-Bcl-2 (1:1000, CST, 2827 S), Bcl-2 (1:1000, ProteinTech, 12789-1-ap), Bax (1:1000, ProteinTech, 50599-2-lg), Cleaved Caspase-3 (1:1000, CST, 9661 S), MST1R (1:1000, ATLAS, HPA008180), and β-Actin (1:1000, Sigma, A5316).

Ding R.B., Chen P., Rajendran B.K., Lyu X., Wang H., Bao J., Zeng J., Hao W., Sun H., Wong A.H., Valecha M.V., Yang E.J., Su S.M., Choi T.K., Liu S., Chan K.I., Yang L.L., Wu J., Miao K., Chen Q., Shim J.S., Xu X, & Deng C.X. (2021). Molecular landscape and subtype-specific therapeutic response of nasopharyngeal carcinoma revealed by integrative pharmacogenomics. Nature Communications, 12, 3046.

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Protein Isolation and Western Blot Analysis

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The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China). Protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells and centrifuged (12,000 r/min) the material at 4 °C for 10 min [26 ]. Protein concentrations were measured on a Thermo Scientific Pierce BCA protein assay kit (Thermo Fisher, Massachusetts, USA) with 1/4 volume of 5 × loading buffer added to the supernatant. A total of 20 μL of protein was blotted using 10% SDS-polyacrylamide gel, then transferred to a polyethylene difluoride (PVDF) membrane (CST, Boston, MA, USA).
After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies (1:1,000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1/SF-1 (Abcam, Cambridge, UK) and against cyclin B, cyclin D, cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, Wuhan, China) were diluted 1:3,000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software Image Lab™, (Bio-Rad, Berkeley, CA, USA).

Shi S., Zhou X., Li J., Zhang L., Hu Y., Li Y., Yang G, & Chu G. (2020). MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells. Journal of Animal Science and Biotechnology, 11, 94.

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Western Blot Analysis of Adipocyte Proteins

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The protocol of western blot was followed according to a previous study [10 (link)]. Briefly, adipocytes were split by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) by adding protease inhibitor (Pierce, WA, USA). The total protein sample was separated in the SDS-polyacrylamide gel. Then, it was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). Next, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. The antibodies Cyclin B, Cyclin D, Cyclin E, PPARγ, and AP2 were purchased from Santa Cruz (CA, USA); C/EBPα and SREBP-1 were purchased from Abcam; p62, LC, ATGL, and HSL were purchased from CST (Boston, MA, USA); and β-tubulin was purchased from Sungene Biotech (Shanghai, China).

Sun Y., Cai R., Wang Y., Zhao R., Qin J, & Pang W. (2020). A Newly Identified LncRNA LncIMF4 Controls Adipogenesis of Porcine Intramuscular Preadipocyte through Attenuating Autophagy to Inhibit Lipolysis. Animals : an Open Access Journal from MDPI, 10(6), 926.

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Apoptosis and Cell Cycle Regulation Analysis

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DMEM, propidium iodide (PI), acridine orange, monodansyl cadaverine, 3-(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT), bafilomycin, chloroquinone (CQ), sodium fluoride, sodium orthovanadate, fetal bovine serum were purchased from Sigma-Aldrich, Missouri, USA. Akt inhibitor IV and VIII, antibodies of caspase-3, cyclin A, cyclin B, cdc2, PARP-1, β-Actin and Akt siRNA were from Santa Cruz Biotechnology, Texas, USA. Antibodies to cdc25, CDK-1, cyclin E were purchased from Piercenet, Illinois, USA. All other antibodies and chemicals were purchased from Cell signaling technology, Massachusetts, USA. Electrophoresis reagents, reagents for protein estimation and protein molecular weight markers were from Bio-Rad Laboratories, California, USA. Polyvinyldifluoride (PVDF) membrane was purchased from Millipore, Massachusetts, USA. K-LISA mTOR activity kit were purchased from Calbiochem, San Diego, USA.

Pathania A.S., Guru S.K., Kumar S., Kumar A., Ahmad M., Bhushan S., Sharma P.R., Mahajan P., Shah B.A., Sharma S., Nargotra A., Vishwakarma R., Korkaya H, & Malik F. (2016). Interplay between cell cycle and autophagy induced by boswellic acid analog. Scientific Reports, 6, 33146.

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