Twenty micrograms of protein was loaded in 4–12% Bis-Tris gels (NuPAGE) and transferred to iBlot nitrocellulose filter (Invitrogen). Primary antibodies: Cyclin A (ab38, 1:500), MYCN (ab16898, 1:250), c-MYC (sc-764, 1:1000), pS62-MYC (ab51156, 1:500), pT58-MYC (ab28842, 1:500), CDK2 (05–596, 1:500, Millipore), p53 (sc-126, 1:1000), cleaved caspase 3 (9661S, 1:500, CST), β-Actin (sc47778, 1:1000), and β-Tubulin (MAB3408, 1:500 and 2146, 1:1000, CST). ECL secondary antibodies (1:5000) (GE healthcare) were detected using Supersignal West Pico Chemiluminescent substrate (ThermoFisher Scientific). Quantification was performed using ImageJ by using the ratio of the sample relative density and the loading control relative density.
Cyclin a
Manufactured by Merck Group
Sourced in Germany
Cyclin A is a protein that plays a critical role in the regulation of the cell cycle. It is involved in the progression of cells through the S phase (DNA replication) and the G2 phase (preparation for cell division) of the cell cycle. Cyclin A functions as a regulatory subunit of cyclin-dependent kinases (CDKs), which are enzymes that control the various stages of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Cyclin e, by Merck Group (3 mentions)
Cyclin d1, by Cell Signaling Technology (3 mentions)
β catenin, by Cell Signaling Technology (3 mentions)
Cyclin b, by Merck Group (3 mentions)
Cyclin e, by Cell Signaling Technology (2 mentions)
Cyclin d, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
C myc, by Merck Group (1 mentions)
Ecl secondary antibodies, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Fzr1 cdh1, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Isolectin gs ib4, by Thermo Fisher Scientific (1 mentions)
Ve cadherin, by Merck Group (1 mentions)
Desmin, by Santa Cruz Biotechnology (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Histone h3, by Abcam (1 mentions)
Pol 2 n 20, by Santa Cruz Biotechnology (1 mentions)
Histone h2b, by Merck Group (1 mentions)
12 protocols using cyclin a
1
Western Blot Analysis of Cell Signaling Proteins
2
Detailed Immunofluorescence Staining Protocol
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Sources and catalogue numbers of antibodies were as follows: Cell Signaling Technology: PTEN (#9559), pS473-Akt (#4060) and pSer240/244-S6 (#2215); BD Pharmingen: p27 (#610242), cyclin-D1 (#556470), BrdU (#347580) and Aurora A Kinase (#610939); NeoMarkers: Ki67 (#RM-9106-S); Abcam: NICD (#ab27526), desmin (ab15200) and PTEN (ab32199); Santa Cruz Biotechnology: VE-cadherin (sc-6458), Geminin (#sc-13015), cyclin-A (#sc-53230), Hes1 (#sc-25392) and Erg (sc-353); Millipore: Plk1 (#06-813) and Fzr1/Cdh1 (#CC43); Sigma-Aldrich: β-actin (A5441) and α-tubulin (T6074). Isolectin GS-IB4 and secondary antibodies conjugated to Alexa 488, Alexa 568 and Alexa 633, and Click-iT EdU Alexa 488 and 647 Imaging Kit were from Molecular Probes. Human (#1506-D4) and mouse Dll4 (#1389-D4) were from R&D Systems. The GDC-0941 compound was from Chem Express Haoyuan (China). The VX680 (MK-0457) compound was from Selleckchem (USA). All chemicals, unless otherwise stated, were from Sigma-Aldrich.
3
Protein Extraction and Western Blotting
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Normal rabbit IgG and rabbit polyclonal antibodies to INTS3, Actin, NABP2, INTS9, INTS11, CUL9, COBRA1 (NELFB), JunB, and HA were obtained from Bethyl Laboratories, Inc. Other rabbit antibodies used include NABP2, NABP1 (Proteintech), Pol II (N-20, Santa Cruz), Spt5 (N-20, Santa Cruz), Histone H2A (Abcam), Histone H3 (Abcam), Histone H2B (Millipore), Histone H4 (gift from CD Allis), INIP (gift from W Wang), Lamin A/C (Cell Signaling), and Cyclin A. The mouse monoclonal Pol II antibody 8WG16 (Millipore) was also used (Supplementary information, Figure S1 ). Cell lysates were generated in lysis buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaF, 0.5% Triton X-100, and either 150 or 250 mM NaCl, as indicated) supplemented with protease and phosphatase inhibitors. CSK (100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, and 10 mM PIPES, pH 6.8) extractions were generated as indicated. Select samples were generated by sonication (Bioruptor; Diagenode) in buffer containing 1 U/μl benzonase. Western blotting was performed as described previously32 (link).
4
Investigating LMO2 Regulation of Angiogenesis
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All chemicals were purchased from Sigma‐Aldrich (St. Louis, MO) unless otherwise stated. EGM‐2 medium and bullet kit was from Lonza (Walkersville, MD). Angiogenesis RT Profiler PCR Array and chromatin immunoprecipitation (ChIP) polymerase chain reaction (PCR) primers were from Qiagen (Valencia, CA). TGF‐β ELISA kits were from R&D Systems (Minneapolis, MN). Taqman primers, cell proliferation kit, AF594‐conjugated acLDL, CellTracker Red, and Griess kit was from Invitrogen (Carlsbad, CA). PE‐human CD31 antibody and APC‐human CD144 antibody was from BD Biosciences (San Jose, CA). ChIP kit was from Cell Signaling Technology (Beverly, MA). Anti‐human LMO2 antibody (clone SP51) was purchased from Spring Bioscience (Pleasanton, CA). Cyclin D1, p21, and horseradish peroxidase (HRP)–conjugated goat anti‐mouse/rabbit antibodies were from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Cyclin A, Cyclin E, CDK2, and CDK4 antibodies were from Millipore (Billerica, MA). β‐Tubulin antibody and anti‐zebrafish lmo2 (zebrafish gene) antibody was from Abcam (Cambridge, UK). Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).
5
Western Blot Analysis of Cellular Proteins
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Whole cellular or tissue proteins were extracted with RIPA lysis buffer (Solarbio, Beijing, China) containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF), according to standard methods. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blot was performed via established protocols 25 (link). Primary antibodies used in this study were as follows: GRHL2 (1:300, Cat. HPA004820, Sigma); cyclin A (1:500, Cat. sc-596), cyclin D1 (1:500, Cat. sc-753), p21 (1:500, Cat. sc-397), p27 (1:500, Cat. sc-528) were from Santa Cruz biotechnology. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit (1:1000, Cat. 7074) and anti-mouse (1:1000, Cat. 7076) from Cell Signaling Technology; Primary antibody against GAPDH (1:5000, Cat. sc-365062, Santa Cruz, USA) was used as a loading control.
6
Immunofluorescence Analysis of Cell Cycle Regulators
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Cultured cells in eight-well chamber slides (Millicell EZ SLIDES) were fixed with cold methanol for 15 min at −20 °C. Then cells were incubated with the following primary antibodies at 4 °C overnight: β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926), and cyclin E (Cell Signaling, 4129). The cells were then incubated with appropriate fluorochrome-conjugated secondary antibodies as described above. Isotype-matched control antibodies were used as negative controls. Nuclei were counterstained with DAPI Staining Solution, and then images were captured using Olympus inverted fluorescence microscope (IX73). For quantitative analysis of mean fluorescence intensity, cells with positive signals in at least six random fields were measured by Olympus cellSens software.
7
Plumbagin Induces Apoptosis in Cervical Cancer
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SiHa and HeLa cervical cancer cell lines were obtained from NCCS, Pune, India. Minimum essential media (MEM)-with glutamine cell culture medium, antibiotic–antimycotic cocktail and other culture reagents were obtained from HiMedia Laboratories, India. Fetal bovine serum (FBS) was from GIBCO, Invitrogen, USA. Plumbagin, NAC, ethylenediaminetetraacetic acid (EDTA), propidium iodide, saponin, bromophenol blue, molecular biology grade DMSO were from Sigma Aldrich, USA, and ethanol was from Merck Biosciences, USA. RNaseA was procured from QIAGEN Hilden, Germany. Antibodies for CDK1, cleaved PARP, cleaved caspase-3, cleaved caspase-9, MMP-2, MMP-9, β-catenin, vimentin, N-cadherin, E-cadherin, peroxidase-conjugated secondary anti-rabbit antibody were from Cell Signaling Technology, USA, secondary anti-mouse antibody, β-actin and cyclin-A were from Sigma Aldrich, and cyclin E, CDK2, Bax, Bcl-2 were from Santa Cruz Biotechnology, USA. PVDF membrane and ECL HRP-linked substrate solution were from Merck Millipore (Billerica, MA). TRIZOL kit for RNA extraction was from Life Technologies, Carlsbad, CA, USA. cDNA synthesis kit was from Takara-Bio, Japan. Other common chemicals and laboratory reagents were from SD Fine-Chem. Ltd and SRL Pvt. Ltd. India.
8
Western Blotting Antibodies Inventory
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Antibodies used for Western blotting were as follows: cyclin A (C4710; Sigma-Aldrich, St. Louis, MO), cyclin B1 (4135; Cell Signaling, Beverly, MA), cyclin D1 (2926; Cell Signaling), p16 (sc-759; Santa Cruz Biotechnology, Dallas, TX), p21 (sc-397; Santa Cruz Biotechnology), p53 (sc-126; Santa Cruz Biotechnology), HMGA2 (sc-30223; Santa Cruz Biotechnology), H3S10phos (ab14955; Abcam, Cambridge, UK), histone H3 (ab1791; Abcam), AURKB (ab2254; Abcam), β-actin (A5441; Sigma-Aldrich), Rb (9309; Cell Signaling), LMNA/C (sc-7292; Santa Cruz Biotechnology), LMNB1 (ab16048; Abcam), IL-6 (MAB2061; R&D Systems, Minneapolis, MN), IL-8 (MAB208, R&D Systems), and MMP3 (a kind gift from Gillian Murphy, University of Cambridge, Cambridge, United Kingdom; Allan et al., 1991 (link)).
9
Western Blot Analysis of Cell Cycle Regulators
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In the presence of RIPA lysis buffer, cells are lysed and total proteins are extracted. Basic experiment procedures of Western blot were consistent with our previous methods [11 (link)]. The introductions of primary antibodies were as follows: SNRNP200 (Cat No. ab176715, Abcam), CyclinA (Cat No. SAB4503499, Sigma-Aldrich), CDK2 (Cat No. 05–363, Merck Millipore), CyclinD (Cat No. 05–137, Merck Millipore), CDK1 (Cat No. 19532-I-AP, Protech), p21WAF1/Cip1 (cyclin-dependent kinase inhibitor 1A), (Cat No. 10355-I-AP, Protech), p53 (Cat No.10442-I-AP, Protech), CyclinB (Cat No.55004-I-AP, Protech) and CyclinE (Cat No. 05–363, Merck Millipore, Darmstadt, Germany). The housekeeping protein we used was Histone H3 (Cat No. Sc-10,809, SantaCruz) or beta-actin (β-actin; Cat. No. ab129348, Abcam, Cambridge, UK). Image J was applied to the quantitative analysis of proteins.
10
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared from cell lines with RIPA lysis buffer kit (Santa Cruz Biotechnology, Santa Cruz, CA), and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Whole-cell proteins (30 μg) were separated on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Corp., Arlington Heights, IL). The membranes were then probed with antibodies against the proteins of H3F3b, BAG-2, Paip2 (Santa Cruz, Santa Cuz, CA), BMI-1 (Millipore, Temecula, CA), EGFR, Bax, and β-actin (Sigma), respectively, for 24 hours. Moreover, membranes were also probed with specific cell cycle, apoptotic and anti-apoptotic antibodies against the following proteins: cyclin A, cyclin B, cyclin D1, p53, caspase 3, caspase 9, NFkb MDM2, BCL2, and BCLxl (Sigma). Washed blots were then incubated with horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz Biotechnology) for one hour at room temperature. Blots were developed using a peroxidase reaction and visualized with the ECL detection system (Bio-Rad,).
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