Cells were re-suspended in sodium dodecyl sulfate (SDS) sample buffer; 106 cells/sample were loaded onto 10% SDS-polyacrylamide gels and electrophoresed. The separated proteins were transferred to nitrocellulose membranes (Bio-Rad) and the membranes were blotted with specific antibodies to cellular and viral proteins. The ZEBRA and BFRF3 antibodies were described previously [38 (link)]. The EA-D (BMRF1) monoclonal antibody (R3.1) was provided by G. Pearson [74 (link)]. GAPDH and FLAG-tagged BGLF3 were detected using mouse monoclonal antibodies (Sigma Aldrich). Antigen-antibody complexes were detected by autoradiography using 125I-protein A or chemiluminescence (GE Healthcare Life Sciences).
Mouse monoclonal antibodies
Manufactured by Merck Group
Sourced in United States
Mouse monoclonal antibodies are laboratory reagents used for a variety of applications, including immunoassays, affinity purification, and cell-based studies. They are produced by fusing mouse B cells with myeloma cells, resulting in a hybridoma cell line that secretes a single, specific antibody. These antibodies are highly specific and can be used to detect or capture target molecules in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab4637, by Abcam (1 mentions)
Ab155668, by Abcam (1 mentions)
H 433, by Santa Cruz Biotechnology (1 mentions)
Anti catalase, by Merck Group (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Rabbit polyclonal antibodies, by Thermo Fisher Scientific (1 mentions)
Ap125, by Progen Biotechnik (1 mentions)
8 protocols using mouse monoclonal antibodies
1
Western Blot Analysis of Viral and Cellular Proteins
2
Antibody Sources for Cell Signaling
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mouse monoclonal antibodies against GST (B-14), Rod (43-K), and CENP-B (F-4; Fig. 1 E and Fig. S4 G) and rabbit polyclonal antibodies against CENP-B (H-65), Cyclin B1 (H-433), His (H-15), and GFP (sc-8334) were obtained from Santa Cruz Biotechnology. Antibodies against Mad1 (ab5783, mouse monoclonal), BubR1 (ab4637, rabbit polyclonal, used for Western blotting in Fig. S3 G), and Chmp4c (ab155668, rabbit polyclonal) were obtained from Abcam. mouse monoclonal antibodies against CENP-A (3–19; GTX13939) and Hec1 (9G3.23; GTX70268) were obtained from Genetex, and mouse monoclonal antibodies against α-tubulin (DM1A) and actin (AC-40) were obtained from Sigma-Aldrich. Anti-pS331 is a rabbit polyclonal antiserum raised against phosphorylated S331 of human Aurora-B (Petsalaki et al., 2011 (link)). A sheep polyclonal antibody against human BubR1 (used for immunofluorescence) was from S. Taylor (University of Manchester, Manchester, England, UK), and rabbit polyclonal antibodies against human ZW10 and Zwilch were from A. Musacchio (Max Planck Institute of Molecular Physiology, Dortmund, Germany). Anti–pChmp4c rabbit polyclonal antiserum against phosphorylated S210, S214, and S215 of human Chmp4c was a gift from P.P. D’Avino (University of Cambridge, Cambridge, England, UK; Capalbo et al., 2012 (link)).
3
Quantifying Antioxidant Enzymes in Vascular Tissue
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The arteries were rapidly frozen and kept at -80°C until needed for protein expression analyses of both catalase and superoxide dismutase in both the control and lead-treated groups. Proteins from homogenized arteries were separated using 12.5 or 10% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and were subsequently incubated overnight at 4°C with anti-catalase (1:100; Sigma Aldrich, St. Louis, MO, USA) and anti-SOD (1:500; Sigma Aldrich, St. Louis, MO, USA) antibodies. Following washing, the membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (1:5000; Stress Gen, Victoria, Canada) conjugated to horseradish peroxidase. Following a thorough washing, the immune complexes were detected using an enhanced horseradish peroxidase/luminal chemiluminescence system (ECL Plus, Amersham International, Little Chalfont, UK) and film (Hyperfilm ECL International). The immunoblot signals were quantified using the ImageJ computer program. The same membrane was used to determine anti-α-actin expression using mouse monoclonal antibodies (1:5000; Sigma, USA).
4
Antibody Labeling of Secretory Granules
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Rabbit polyclonal antibodies directed against AnxA2 (p36) purified from bovine aorta were diluted 1:200 (a gift from J.C. Cavadore, Institut National de la Santé et de la Recherche Médicale U-249, Paris, France). Rabbit polyclonal antibodies directed against DBH (EC.1.14.17.1) purified from bovine chromaffin granules were diluted 1:75 to specifically label secretory granules in chromaffin cells (Chasserot-Golaz et al., 1996 (link)). Mouse monoclonal antibodies directed against syntaxin, TRITC-conjugated phalloidin, and Latrunculin B were from Sigma-Aldrich. Mouse monoclonal antibodies directed against GFP (clones 7.1 and 13.1) were from Roche. Secondary goat anti–rabbit and anti–mouse antibodies and streptavidin coupled to Alexa Fluor conjugates (488, 555, or 647) or gold particles were from Molecular Probes (Invitrogen) and Aurion, respectively. Fluorescent cholera toxin B subunit coupled to Alexa Fluor 633 or 555 were from Molecular Probes (Thermo Fisher Scientific), and WA was from Calbiochem inhibitors (EMD Millipore).
5
Immunofluorescence Localization of Cytoskeletal Proteins
6
Oligodendrocyte Progenitor Cell Markers
7
Rac1 and Cdc42 Activation Assay
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The assay was performed as previously described52 (link). Briefly, cells were chilled on ice and lysed in ice-cold buffer containing 0.5% Triton X-100, 25 mM HEPES (pH 7.3), 150 mM NaCl, 4% glycerol, 0.1 mM AEBSF, 4 µg/ml aprotinin and 5 mM DTT. Lysates were centrifuged for 6 min at 16,000×g. Supernatants were immediately frozen in liquid nitrogen and stored at –80 °C until use. An aliquot of each supernatant collected before freezing was denatured in SDS-PAGE lysis buffer to measure the total Rac1 and Cdc42 content by Western blotting with mouse monoclonal antibodies (from Millipore (1/2000; 05-389) and BD Biosciences (1/500; 610929), respectively). For pull-down assays, supernatants were incubated for 30 min at 4 °C with 30 µg of GST-PBD protein containing the Cdc42-binding and Rac-binding region of PAK-1B linked to glutathione-Sepharose beads. The beads were washed four times in lysis buffer and boiled in 60 µl of SDS-PAGE lysis buffer. For loading control, samples were incubated for 3 h at RT with a mouse anti-human GAPDH antibody (1/2000; Millipore).
8
Comprehensive Antibody Panel for Perilipin Proteins
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Guinea pig polyclonal antibodies against perilipin 1 (GP29; dilution 1:1000), perilipin 2 (GP40; dilution 1:1000), perilipin 3 (GP30; dilution 1:1000), perilipin 4 (GP38; dilution 1:1000), and perilipin 5 (GP31; dilution 1:1000) were from Progen Biotechnik (Heidelberg, Germany). mouse monoclonal antibodies against perilipin 1 (Peri 112.17; dilution 1:100) and perilipin 2 (clone AP125; dilution 1:100) were from Progen Biotechnik, and mouse monoclonal antibodies against actin (C4; dilution 1:10.000) were from Millipore (Billerica, MA). In addition, rabbit polyclonal antibodies against N-terminal amino acid 10 through 29 (Plin5 1.1 and 1.2; dilution 1:100) and against central amino acid 254 through 296 (Plin5 2.1 and 2.2; dilution 1:100) of perilipin 5 were generated by Thermo Fisher Scientific (Bremen, Germany 29 ).
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