Anti cyclin b1

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Anti-cyclin B1 is a laboratory reagent that can be used to detect and study the expression of cyclin B1 in biological samples. Cyclin B1 is a key regulator of the cell cycle and plays a crucial role in the progression from the G2 phase to mitosis. The Anti-cyclin B1 product can be used in various research applications, such as immunoblotting, immunoprecipitation, and immunohistochemistry, to investigate the levels and localization of cyclin B1 in cells and tissues.

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Cyclin B1 (162 citations) Mouse anti-cyclin B1 (8 citations) Anti-cyclin B1 (sc-245, (5 citations) Anti-Cyclin-B1 antibody (5 citations) Rabbit anti-cyclin B1 (4 citations) Mouse monoclonal anti-cyclin B1 (3 citations) Anti-cyclin B1 (H 433) (2 citations)

69 protocols using anti cyclin b1

Immunoprecipitation and Western Blotting

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Collected cells were washed with cold PBS and lysed in ice-cold buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40] supplemented with complete protease inhibitors (Sigma-Aldrich) on ice for 10 min. The samples were centrifuged at 13,000rpm for 10 min. The lysates were aliquoted into two tubes and incubated with either the designated antibody or an appropriate IgG control for 16 hours at 4 °C. Protein A/G agarose (Invitrogen) was used to precipitate antibody-protein complexes.
For Western blotting, anti-CD24 (Santa Cruz Biotechnology, 1:1000), anti-p27 (Cell Signaling, 1:1000), anti-p21 (Cell Signaling, 1:1000), anti-p53 (Santa Cruz Biotechnology, 1:2000), anti-Lamin (Santa Cruz Biotechnology, 1:5000), anti-H1.5 (Abcam, 1:5000), anti-Cyclin B1 (Santa Cruz Biotechnology, 1:3000), anti-GFP (Cell Signaling, 1:2000), anti-NPM1 (Abcam, 1:3000), anti-HA (Sigma-Aldrich, 1:3000), anti-Myc (Abcam, 1:3000), anti-ARF (Santa Cruz Biotechnology, 1:1000), anti-MDM2 (Santa Cruz Biotechnology, 1:1000), and anti-β-actin (Sigma, 1:5000) antibodies were used as primary antibodies. The membranes were incubated for 1 hour at room temperature in 0.25% nonfat milk with a 1:3,000–5,000 dilution of either anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (Cell Signaling).

Wang L., Liu R., Ye P., Wong C., Chen G.Y., Zhou P., Sakabe K., Zheng X., Wu W., Zhang P., Jiang T., Bassetti M.F., Jube S., Sun Y., Zhang Y., Zheng P, & Liu Y. (2015). Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation. Nature Communications, 6, 5909.

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Western Blot Analysis of Cell Cycle Regulators

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Viable cells in PBS buffer or cell lysates in RIPA buffer were reduced with DTT in 2x Laemmli buffer and denatured at 100°C. Western blotting was carried out as previously described [41 (link)]. Target proteins were detected using anti-α-tubulin (1:10000, T5168, Sigma‒Aldrich, St. Louis, MO, USA), anti-TRMT112 (1:500, sc-398481, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin B1 (1:500, sc-245, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin E (1:500, sc-247, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin A (1:500, sc-271682, Santa Cruz Biotechnology, Dallas, TX, USA), anti-N6AMT1 (1:1000, CQA1550, Cohesion Biosciences, London, UK), and anti-GAPDH (1:2000, sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Goat anti-rabbit-HRP (1:10000, 31460, Invitrogen, Carlsbad, CA, USA) and goat anti-mouse-HRP (1:10000, 31430, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.

Mutso M., Brūmele B., Serova E., Väärtnõu F., Suija M, & Kurg R. (2024). The methyltransferase N6AMT1 participates in the cell cycle by regulating cyclin E levels. PLOS ONE, 19(2), e0298884.

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Western Blot Antibodies and Imaging

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The primary antibodies used in the western blot analyses include anti-BRD4N (targeting BRD4 156–284, 1:40,000), anti-BRD4C (targeting BRD4 1313–1362, 1:40,000), anti-Cyclin B1 (1:500, sc-245, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GAPDH (1:4000, G8140-01, US Biological, Salem, MA, USA), anti-Actin (MAB1501, Millipore, Burlington, MA, USA) and anti-HA-HRP (1:2000, 12013819001, Roche, Rotkreuz, Switzerland). HRP-linked anti-rabbit IgG (1:3000; 7074S; Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:3000; 7076S; Cell Signaling Technology) were used as secondary antibodies. Western blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and images were captured using an Amersham Imager 600 (GE Healthcare). Detailed information about western blot can be found in Figures S6 and S7.

Wang R., Yang J.F., Ho F., Robertson E.S, & You J. (2020). Bromodomain-Containing Protein BRD4 Is Hyperphosphorylated in Mitosis. Cancers, 12(6), 1637.

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Comprehensive Immunoblotting Methodology

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Immunoblotting was performed as described previously (35 (link)). The following antibodies were used at a concentration of 0.1 to 0.5 μg/ml: anti-pMCM2 (3378-1; Epitomics Inc.), anti-MCM2 (sc-9839; Santa Cruz Biotechnology), anti–pPOL II (04-1571; Millipore), anti–POL II (05-952; Millipore), anti-Lamin B1 (ab16048; Abcam), anti-FANCD2 (sc-20022; Santa Cruz Biotechnology), anti-pCDC6 (ab76422; Abcam), anti-CDC6 (ab109315; Abcam), pChk1 (#2348; Cell Signaling Technology), anti-Chk1 (#2345; Cell Signaling Technology), anti-pCDK1 (#9111; Cell Signaling Technology), anti-CDK1 (sc-954; Santa Cruz Biotechnology), anti-gH2AX (#2577; Cell Signaling Technology), anti-PARP1 (#9542; Cell Signaling Technology), anti–cyclin B1 (sc-752; Santa Cruz Biotechnology), anti-CDC7 (sc-56274; Santa Cruz Biotechnology), anti-DBF4 (ab124707; Abcam), and anti-GAPDH (MAB374; Chemicon). Immunoblotted proteins were visualized by chemiluminescence.

Iwai K., Nambu T., Dairiki R., Ohori M., Yu J., Burke K., Gotou M., Yamamoto Y., Ebara S., Shibata S., Hibino R., Nishizawa S., Miyazaki T., Homma M., Oguro Y., Imada T., Cho N., Uchiyama N., Kogame A., Takeuchi T., Kurasawa O., Yamanaka K., Niu H, & Ohashi A. (2019). Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor. Science Advances, 5(5), eaav3660.

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Western Blot Analysis of Protein Expression

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Anti-FLAG and anti-beta actin (Sigma-Aldrich, USA); anti-GNL1, anti-RPS20, anti-CDK1 and anti-MDM2 (Abcam, UK); anti-GFP, anti-Cyclin B1 and anti-p53 (Santa Cruz, USA); anti-p21, anti-Cyclin D1, anti-CDK4, anti-pRbSer-780, anti-Rb and anti-E2F1 (Cell Signaling Technology Inc., USA) antibodies were used in western blot analysis for checking protein expression.

Krishnan R., Boddapati N, & Mahalingam S. (2018). Interplay between human nucleolar GNL1 and RPS20 is critical to modulate cell proliferation. Scientific Reports, 8, 11421.

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Mitochondrial Dynamics and Cell Cycle Analysis

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MitoTracker Green FM, DAPI, and Alexa Fluor 488– phalloidin were purchased from Life Technologies (Carlsbad, CA). Digitonin and etoposide were from Wako Pure Chemical Industries (Osaka, Japan). Mdivi-1 was from Enzo Life Sciences (Farmingdale, NY). Z-VAD-FMK was purchased from Peptide Institute (Osaka, Japan). NU6140 and BI2536 were from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used for Western blotting and immunostaining: anti-Drp1, anti–cytochrome c (BD Biosciences, Billerica, MA), anti–γ-tubulin (Sigma-Aldrich, St. Louis, MO), anti-Smac (ProSci, Poway, CA), anti–phospho-Plk1 (Thr-210), anti-Plk1 (Abcam, Cambridge, United Kingdom), anti–phospho-CDK2 (Thr-160), anti-γ-H2AX, anti–phospho-histone H3 (Ser-10; Cell Signaling Technology, Beverly, MA), Alexa Fluor 488 anti-mouse immunoglobulin G (Life Technologies), anti–voltage-dependent anion-selective channel protein 1 (VDAC1), anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-CDK2, anti–cyclin A, anti–cyclin E, anti–cyclin B1, anti-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). The Western Lightning Plus-ECL chemiluminescence detection kit was purchased from PerkinElmer-Cetus (Boston, MA).

Yamamori T., Ike S., Bo T., Sasagawa T., Sakai Y., Suzuki M., Yamamoto K., Nagane M., Yasui H, & Inanami O. (2015). Inhibition of the mitochondrial fission protein dynamin-related protein 1 (Drp1) impairs mitochondrial fission and mitotic catastrophe after x-irradiation. Molecular Biology of the Cell, 26(25), 4607-4617.

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Immunoprecipitation and Immunoblotting Protocol

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Cells were harvested and washed twice with cold PBS. Collected cells were lysed with NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris [pH 8], 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Sigma-Aldrich). For the SUMOylation assay, 1% SDS and 20 mM NEM were also added. Next, cell lysates were sonicated and then cleared using centrifugation (4°C, 20,000 g, 10 min, twice). The supernatant was then incubated with an antibody/bead conjugate at 4°C for 4 h. Immunoprecipitates were then subjected to immunoblot analysis with the indicated antibodies. The following antibodies were used in the study: anti-PLK1 (Thermo Fisher Scientific), anti-Flag (Sigma-Aldrich), anti–β-actin (Sigma-Aldrich), anti-Ubc9 (Abcam), anti-Myc (BD), anti-HA (Vandenise), anti-T7 (Novagen), anti-SUMO-1 (Invitrogen), anti-His (Cell Signaling), anti-α-tubulin (Sigma-Aldrich), anti-ubiquitin (Upstate), anti-lamin A/C (Assay Biotech), anti-GAPDH (Santa Cruz Biotechnology), anti-cdh1 (Santa Cruz Biotechnology), anti-cyclin B1 (Santa Cruz Biotechnology), and anti-KLHL22 (Proteintech).

Wen D., Wu J., Wang L, & Fu Z. (2017). SUMOylation Promotes Nuclear Import and Stabilization of Polo-Like Kinase 1 to Support its Mitotic Function. Cell reports, 21(8), 2147-2159.

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Protein Extraction and Analysis from Cultured Cells

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Cells growing in Petri dishes were collected and lysed in cell lysis buffer (Cell Signaling Technology, Boston MA USA) containing 20 mmol/L Tris-HCL (PH7.5), 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGDA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO4, 1 μg/ml leupeptin, and 1 mmol/l phenylmethane sulphonyl fluoride. Membranes were incubated overnight at 4°C with specific antibodies in TBS-T (TBS-0.1% Tween20) and subsequently for one hour with the appropriate horseradish-peroxidase-conjugated anti-rabbit/mouse secondary antibody. The following mouse monoclonal antibodies were used: Anti-CyclinB1 (Santa Cruz Biotechnology, Dallas Texas USA), anti-Caspase-8, anti-β-actin (both from Cell Signaling Technology, Boston MA USA). The following rabbit polyclonal antibodies were used: anti-Caspase-3, anti-PARP, anti-Bid, anti-Bad, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Mcl-1 (all from Cell Signaling Technology), anti-c-MET, anti-phosphorylated c-MET (Tyr1349 and Tyr1234/1235). For Caspase-3/7 activity assays the Apo-ONE® Reagent kit (Promega, Madison WI USA) was used according to the instructions of the manufacturer. Actin was used for control of appropriate protein load for each membrane. In figures, one actin loading control has been exemplarily shown.

Lu S., Török H.P., Gallmeier E., Kolligs F.T., Rizzani A., Arena S., Göke B., Gerbes A.L, & De Toni E.N. (2015). Tivantinib (ARQ 197) affects the apoptotic and proliferative machinery downstream of c-MET: role of Mcl-1, Bcl-xl and Cyclin B1. Oncotarget, 6(26), 22167-22178.

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Investigating UCHL1 and Cell Cycle Regulation

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Anti-cyclin B1 from Santa Cruz (sc-245) was used for immunohistochemistry, western blot, immunoprecipitation, immunofluorescence and the proximity ligation assay. Anti-UCHL1 from Sigma-Aldrich (HPA005993) was used for immunohistochemistry, immunofluorescence and proximity ligation assay; Anti-UCHL1 from Cell Signaling Technology (11896) was used for western blot; Anti-UCHL1 from R&D Systems (MAB6007) was used for immunoprecipitation. Additional antibodies used for western blot were anti-cyclin D1 (sc-753, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-cyclin E (sc-481, Santa Cruz Biotechnology Inc.), anti-p53 (48818, Cell Signaling Technology, Danvers, MA, USA), anti-ubiquitin (3936, Cell Signaling Technology), anti-β-actin (4967, Cell Signaling Technology), anti-β-catenin (9562, Cell Signaling Technology), anti-GSK3α/β (5676, Cell Signaling Technology), anti-p21 (sc-397, Santa Cruz Biotechnology Inc.), and anti-p27 (sc-529, Santa Cruz Biotechnology Inc.).
UCHL1 was transiently silenced by transfection with Silencer select siRNAs (s14616 and s14618; Life Technologies, Carlsbad, CA, USA) duplexed with Lipofectamine RNAiMAX (Life Technologies) at a final concentration of 5 nM. Non-targeting Silencer Select siRNA was the negative control (Life Technologies).

Kwan S.Y., Au-Yeung C.L., Yeung T.L., Rynne-Vidal A., Wong K.K., Risinger J.I., Lin H.K., Schmandt R.E., Yates M.S., Mok S.C, & Lu K.H. (2020). Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression. Cancers, 12(1), 118.

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Western Blot Analysis of Sertoli Cells

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Human Sertoli cells with miR-133b mimics or inhibitor treatment were lysed with RIPA buffer (Santa Cruz) for 30 min on ice. After 30 min of lysis, cell lysates were cleared by centrifugation at 12,000 g for 20 min, and the concentrations of proteins were measured by BCA kit (Dingguo Company, catalog no: P0012). Thirty micrograms of cell lysate from each sample were used for SDS-PAGE (Bio-Rad Laboratories), and Western blots were performed according to the protocol as described previously [8 (link)]. The chosen antibodies included anti-GLI3 (Sigma, catalog no: WH0002737M1, dilution: 1:500), anti-Cyclin B1 (Santa Cruz, catalog no: SC-752, dilution: 1:200), anti-Cyclin D1 (Santa Cruz, catalog no: SC-717, dilution: 1:200), anti-PCNA (Santa Cruz, catalog no: SC-7907, dilution: 1:200), and anti-ACTB (Protein tech, catalog no: HRP-60008, dilution: 1:5000). After extensive washes with TBST, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad).

Yao C., Sun M., Yuan Q., Niu M., Chen Z., Hou J., Wang H., Wen L., Liu Y., Li Z, & He Z. (2016). MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3. Oncotarget, 7(3), 2201-2219.

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