Anti ccnb1

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-CCNB1 is a laboratory reagent that detects the protein CCNB1 (Cyclin B1). CCNB1 is a key regulator of the cell cycle and plays a critical role in the G2/M transition. This antibody can be used to monitor CCNB1 expression and activity in various experimental systems.

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CCNB1 (14 citations)

14 protocols using anti ccnb1

Immunoblot Analysis of Circadian Clock and Cell Cycle Proteins

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Immunoblot analysis for U2OS cells was performed as described [85 (link)] using the following antibodies: anti-BMAL1 (14020, Cell Signaling, Danvers, MA), CRY1 (A302-614A, Bethyl Laboratories, Montgomery, TX), anti-CRY2 (13997-1-AP, Proteintech, Chicago, IL), anti-RB (9309, Cell Signaling, Danvers, MA), anti-pRB-S807/811(8516, Cell Signaling, Danvers, MA), anti-pRB-S795 (9301, Cell Signaling, Danvers, MA), anti-pRB-S780 (8180, Cell Signaling, Danvers, MA), anti-pRB-S612 (AP3236a, Abgent, San Diego, CA), anti-CCNB1 (#4138, Cell Signaling, Danvers, MA), anti-cyclin D1 (ab134175, Abcam, Cambridge, MA), anti-CDK4 (12790, Cell Signaling, Danvers, MA), anti-CDK6 (14052-1-AP, Proteintech, Chicago, IL). For liver tissue extracts, anti-CDK4 (ab137675, Abcam, Cambridge, MA), anti-RB (ab24, Abcam, Cambridge, MA), and anti-pRB-S807/811 (ABC132, MilliporeSigma, Burlington, MA) were used. Anti-GAPDH (sc25778, Santa Cruz Biotech, Dallas, TX) was used as loading control antibody for both U2OS cell and liver tissue extracts.

Lee Y., Lahens N.F., Zhang S., Bedont J., Field J.M, & Sehgal A. (2019). G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment. PLoS Biology, 17(4), e3000228.

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Regulation of Cell Cycle by USP22

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Human colon cancer HCT116 cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum. usp22^+/+ and usp22^−/− mouse embryonic fibroblasts were isolated and used as described [20 (link)]. USP22 and its mutant plasmids were used as described [20 (link)] or cloned into pcDNA3.1. Plasmid DNA that expresses CDC20, CDH1, FBW7 and USP10 were purchased from Addgene company. Plasmids of CCNB1, APC4, APC5 and APC8 are made by PCR and then cloned into PCMV vector. Antibodies and their sources used in this study included anti-USP22 (Sigma Aldrich, St Louis, MO, USA) or as described [16 (link)], anti-CCNB1 (for immunohistochemistry), anti-CCNA, anti-CCNE, CDC20, APC8 (Cell Signaling Technology, Danvers, MA, USA), anti-Flag (Sigma Aldrich), anti-Myc, anti-HA, and anti-CCNB1 (for western blotting; Santa Cruz, Inc., Santa Cruz, CA, USA). Anti-phospho-Thr/Ser antibody was purchased from Abcam. Small interfering RNA/shRNA that specifically knocks down USP22 and control small interfering RNA/shRNA were purchased from Open Biosystem company.

Lin Z., Tan C., Qiu Q., Kong S., Yang H., Zhao F., Liu Z., Li J., Kong Q., Gao B., Barrett T., Yang G.Y., Zhang J, & Fang D. (2015). Ubiquitin-specific protease 22 is a deubiquitinase of CCNB1. Cell Discovery, 1, 15028-.

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IGF2BP3 Expression Analysis via qPCR and Western Blot

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RNA isolation and RT–qPCR analysis were carried out according to previous studies (20 (link)). β-actin served as an internal control. The sequences of the primers used in the experiment are as follows. Human IGF2BP3: 5′- TCGAGGCGCTTTCAGGTAAA-3′ (forward), 5′- AAACTATCCAGCACCTCCCAC-3′ (reverse). Mouse Igf2bp3: 5′- CCTGGTGAAGACGGGCTAC-3′ (forward), 5′- TCAACTTCCATCGGTTTCCCA-3′ (reverse).
Protein extraction and Western blot analysis were carried out according to previous studies (20 (link)). The primary antibodies included rabbit anti-IGF2BP3 (1:1000, Proteintech, Chicago, USA), anti-CCNB1 (1:1000, Shanghai, China) and anti-C-Myc (1:2,000, Cell Signaling Technology, Beverly, MA, USA). Band densities on autoradiograms were densitometrically quantified (Quantity One software; Bio-Rad), with GAPDH serving as the internal control.

Geng Q., Cao X., Fan D., Gu X., Zhang Q., Zhang M., Wang Z., Deng T, & Xiao C. (2022). Diagnostic gene signatures and aberrant pathway activation based on m6A methylation regulators in rheumatoid arthritis. Frontiers in Immunology, 13, 1041284.

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Protein Expression Analysis of Cell Lysates

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Lysates of G9pCDH cells and G98 cells were generated with 8 M urea buffer (8 M urea, 1% Triton 100, 50 mM Tris-base ph = 7.5, 100 mM NaCl, 1 mM EGTA, 1 mM RDTA, 50 mM beta-glycerophosphate), supplemented with protease inhibitors (Millipore-Sigma #539134) and phosphatase inhibitors (Millipore-Sigma/Roche #4906845001) after treatment with CAN-2409, AdVtk (MOI 500) or mock for 48 h. Protein concentration was determined with Bradford assay. Primary antibodies used were anti-CCNB1 (Cell Signaling Technologies #12231), anti-PLK1 (Cell Signaling Technologies, #4513), anti-c-MYC (Cell Signaling Technologies #5605) and Alexa Fluor 488-labeled anti-GAPDH (#sc-365,062, Santa Cruz), anti-rabbit (Cell Signaling Technologies #7074) was used a as secondary antibody. Band signal intensity quantification was carried out with Bio-Rad Image Lab software in triple measurements.

Koch M.S., Zdioruk M., Nowicki M.O., Hoetker M.S., Herbert Z.T., Barone F., Tak P.P., Chiocca E.A., Tabatabai G, & Lawler S.E. (2023). Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models. Frontiers in Medicine, 10, 1140352.

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Western Blot Antibody Validation

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Antibodies used in the present study were: anti-HA (#SC-7392; mouse monoclonal, dilution 1:3000); anti-AKT1 (#SC-5298; mouse monoclonal, dilution 1:1000) and anti-GAPDH (#SC-25778; rabbit polyclonal, dilution 1:1000) from Santa Cruz; anti-CDC2 (#77055; rabbit polyclonal, dilution 1:1000); anti-CCNB1 (#4138; rabbit polyclonal, dilution 1:1000) from Cell Signalling Technology; anti-BUB3 (#ab133699; rabbit monoclonal, dilution 1:10000); anti-API5 (#ab56392; mouse monoclonal, dilution 1:1000) and anti-SH3PX1 (#EPR14399; rabbit monoclonal, dilution 1:2000) from Abcam; secondary antibodies were procured from Licor Biosciences-Odyssey goat anti-mouse (#926-32210, dilution 1:15000) and Odyssey goat anti-rabbit (#926-32211, dilution 1:15000).

Duggal S., Jailkhani N., Midha M.K., Agrawal N., Rao K.V, & Kumar A. (2018). Defining the Akt1 interactome and its role in regulating the cell cycle. Scientific Reports, 8, 1303.

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Comprehensive Immunoblotting Analysis of Cell Signaling

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Immunoblotting was performed with standard procedure. The following antibodies were used in this study: anti-NANOG (#8822, Cell Signaling Technology, MA, USA), anti-TFAP2C (sc-12762, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (#5174, Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 (#9664, Cell Signaling Technology, MA, USA), anti-cleaved PARP1 (ab32064, abcam), anti-CDH1 (#14472, Cell Signaling Technology, MA, USA), anti-β-CATENIN (#8480, Cell Signaling Technology, MA, USA), anti-PCNA (#13110, Cell Signaling Technology, MA, USA), anti-CCND2 (#3741, Cell Signaling Technology, MA, USA), anti-CCNA2 (#91500, Cell Signaling Technology, MA, USA), anti-CCNB1 (#4135, Cell Signaling Technology, MA, USA), anti-CDK1 (#9116, Cell Signaling Technology, MA, USA), anti-CDK2 (#2546, Cell Signaling Technology, MA, USA), anti- CCND1 (#55506, Cell Signaling Technology, MA, USA), and anti-EPCAM (#93790, Cell Signaling Technology, MA, USA). Immunoblots were visualized on iBright CL1000 Imaging Systems (Thermo Fisher Scientific).

Wang Y., Chen S., Jiang Q., Deng J., Cheng F., Lin Y., Cheng L., Ye Y., Chen X., Yao Y., Zhang X., Shi G., Dai L., Su X., Peng Y, & Deng H. (2020). TFAP2C facilitates somatic cell reprogramming by inhibiting c-Myc-dependent apoptosis and promoting mesenchymal-to-epithelial transition. Cell Death & Disease, 11(6), 482.

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Investigating USP22 Regulation in Colon Cancer

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Human colon cancer HCT116 cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum. usp22^+/+ and usp22^−/− mouse embryonic fibroblasts were isolated and used as described [20 (link)]. USP22 and its mutant plasmids were used as described [20 (link)] or cloned into pcDNA3.1. Plasmid DNA that expresses CDC20, CDH1, FBW7 and USP10 were purchased from Addgene company. Plasmids of CCNB1, APC4, APC5 and APC8 are made by PCR and then cloned into PCMV vector. Antibodies and their sources used in this study included anti-USP22 (Sigma Aldrich, St Louis, MO, USA) or as described [16 (link)], anti-CCNB1 (for immunohistochemistry), anti-CCNA, anti-CCNE, CDC20, APC8 (Cell Signaling Technology, Danvers, MA, USA), anti-Flag (Sigma Aldrich), anti-Myc, anti-HA, and anti-CCNB1 (for western blotting; Santa Cruz, Inc., Santa Cruz, CA, USA). Anti-phospho-Thr/Ser antibody was purchased from Abcam. Small interfering RNA/shRNA that specifically knocks down USP22 and control small interfering RNA/shRNA were purchased from Open Biosystem company.

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Immunoblotting of Signaling Pathways

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Immunoblotting was performed as described previously^{26 (link),27 (link)}. Briefly, cells were cultured in 100-mm dishes at a density of 5 × 10⁵ cells per dish and treated with 5 μM gefitinib and/or 100 nM everolimus for 24 h or 48 h. Twenty micrograms of each protein samples from cell lysates were separated via 7–12% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (GE Healthcare, Westborough, MA, US). Blots were blocked and incubated overnight at 4 °C with the following primary antibodies: anti-phospho-ERK1/2 (Thr 202, Tyr 204), anti-ERK1/2, anti-phospho-AKT (Ser 473), anti-AKT, anti-phospho-mTOR (Ser 2448), anti-mTOR, anti-phospho-P70S6K (Thr 389), anti-P70S6K, anti-phospho-4E-BP1 (Thr 37, Thr 46), anti-4E-BP1, anti-EGFR, anti-IGFR, anti-CCNE1, anti-CCNB1 and anti-CDKN1C at final dilutions of 1:1000 (Cell Signalling Technology, Danvers, MA, USA) and anti-β-actin at final dilution of 1:5000 (Calbiochem, San Diego, CA, USA). Western blots signals were visualized using a chemiluminescence kit (Amersham Bioscience, Piscataway, NJ, USA) and quantified using ImageJ software. The intensity of individual bands was expressed relative to the control signal.

El Guerrab A., Bamdad M., Bignon Y.J., Penault-Llorca F, & Aubel C. (2020). Co-targeting EGFR and mTOR with gefitinib and everolimus in triple-negative breast cancer cells. Scientific Reports, 10, 6367.

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Western Blot for Cell Cycle Regulators

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The same protein lysates used for RPPA were also used for Western Blot analysis. Briefly, 30μg of protein was fractionated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with blocking solution (5% Milk in TBS-T) for 1 hour at room temperature, and then blotted with relevant antibody (anti-p21; anti-p27 and anti-CCNB1 from Cell Signaling) diluted on a BSA 1% solution overnight at 4°C in the concentration of 1:1000. HRP-conjugated secondary antibody was detected by using the Enhanced Chemiluminescence Kit (GE Healthcare).

Goedert L., Pereira C.G., Roszik J., Plaça J.R., Cardoso C., Chen G., Deng W., Yennu-Nanda V.G., Silva WA J.r., Davies M.A, & Espreafico E.M. (2016). RMEL3, a novel BRAFV600E-associated long noncoding RNA, is required for MAPK and PI3K signaling in melanoma. Oncotarget, 7(24), 36711-36718.

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Western Blotting for Protein Expression

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Cells were lysed with RIPA buffer (Beyotime, Beijing, China) and the extracted protein was quantified using bicinchoninic acid (Beyotime, Beijing, China). Protein samples (15 μg total protein per lane) were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were incubated with high-affinity anti-MYBL2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin (1:1000, Abcam, Cambridge, MA, USA), anti-Vimentin (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-CCNB1 (1:1000, Cell Signaling Technology), anti-P21 (1:1000, Cell Signaling Technology) and anti-GAPDH (1:2000, Cell Signaling Technology) antibodies, overnight at 4°C. Then, after washing, the membranes were incubated with HRP-conjugated secondary monoclonal antibody (1:5000, Cell Signaling Technology) at room temperature for 1 hour. The blots were developed using a chemiluminescence system (Bio-Rad) and the protein bands were quantified.

Li M., Liu Y., Liu J., Li W., Li N., Xue D., Zhang X, & Wang P. (2019). Circ_0006332 promotes growth and progression of bladder cancer by modulating MYBL2 expression via miR-143. Aging (Albany NY), 11(22), 10626-10643.

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