U2OS cells grown in 60-mm dishes were transfected with 1 μg Myc-MdmX and 6 μg wild-type Mdm2 or Mdm2 mutant plasmid constructs using Lipofectamine 2000 transfection reagent (Invitrogen) and DMEM was changed 4 h later. Each transfection mixture also contained 0.1 μg pEGFP-N1 as a control for transfection efficiency. The empty plasmid pcDNA3.1 was used to bring the total amount of transfected DNA to 7.1 μg in cells transfected with MdmX expression plasmid alone. Cells were cultured for 30 h, washed with cold PBS, and lysed with 0.3 ml of SDS-PAGE sample buffer. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-Myc 9E10 (Merck, MABE282, 1:1,000), anti-Mdm2 Ab-1 (Merck, OP46, 1:1,000), and anti-GFP 7.1/13.1 (Roche, 11814460, 1:5,000) antibodies.
Mabe282
Manufactured by Merck Group
MABE282 is a compact centrifuge designed for routine laboratory applications. It features a brushless motor, microprocessor control, and a compact footprint. The centrifuge can accommodate a variety of rotor configurations and sample volumes.
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Lab products found in correlation
F3165, by Merck Group (1 mentions)
Goat anti mouse igg hrp antibody, by Bio-Rad (1 mentions)
Complete mini, by Roche (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by Merck Group (1 mentions)
Sc 789, by Santa Cruz Biotechnology (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Merck Group (1 mentions)
Ab4441, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Ro 3306, by Merck Group (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
T9026, by Merck Group (1 mentions)
Ab2811, by Abcam (1 mentions)
Zm447439, by Selleck Chemicals (1 mentions)
I1784, by Merck Group (1 mentions)
Purvalanol a, by Merck Group (1 mentions)
Bi2536, by Axon Medchem (1 mentions)
5 protocols using mabe282
1
Analyzing Mdm2 and MdmX Interaction
2
Rabies Virus Protein Expression Analysis
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Transfection with plasmids that expressed RABV proteins was performed using Metafectane reagent, following the manufacturers’ instructions (Biontex). We added 500 nM 17-AAG to the transfected cells 6 h after infection, and the incubation was terminated 48 h after transfection. The protein extracts prepared with the RIPA buffer were resolved on 10% PAGE-SDS gels to detect myc-tagged G, M, N, and P proteins. 3xFLAG-L protein was resolved on 8% PAGE-SDS gel. The insoluble protein was separated by centrifugation for 20 min at 14,000× g and the pellet was solubilized in 8M urea before electrophoresis. The proteins were detected using monoclonal antibodies specific for Flag (Sigma, F3165) and myc (Merck, MABE282). A goat anti-mouse IgG-HRP antibody was obtained from Bio-Rad (cat. no. 170-6516). The original pictures were taken with a CCD camera with the exposure time adjusted to avoid pixel saturation. The registered signals were within the linear response range of the camera.
3
Analyzing Mdm2 and MdmX Interactions
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HEK293 cells were transfected with 6 μg DNA per 100-mm plate using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with a proteasome inhibitor (10 μM MG132 in cell culture medium) 24–36 h post-transfection, 4 h later washed with PBS, and lysed in Triton X-100 lysis buffer (1% Triton X-100, 150 mM NaCl, and 50 mM Tris pH 8.0) containing protease inhibitors (Complete Mini, Roche). Lysates were pre-cleared with 50 μl of protein G-Sepharose (Millipore). Immunoprecipitations were performed at 4°C with 1–2 μg of an antibody bound to 50 μl of protein G-Sepharose for 1 h. Mdm2 was immunoprecipitated with anti-Mdm2 antibody IF2 (Ab-1, Merck, OP46) and Myc-MdmX with anti-Myc antibody 9E10 (Merck, MABE282). Immunoprecipitated proteins were washed with lysis buffer and resuspended in SDS-PAGE sample buffer. Proteins from whole-cell extracts and immunoprecipitations were resolved by SDS-PAGE and analyzed by Western blotting with anti-Mdm2 Ab-1 (Merck, OP46, 1:1,000) and anti-Myc 9E10 (Merck, MABE282, 1:1,000) or anti-Myc A-14 (Santa Cruz Biotechnology, sc-789, 1:1,000).
4
Western Blot Analysis of Epigenetic Markers
5
Antibody Validation for Nucleocytoplasmic Transport
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The following antibodies were used for immunofluorescence (IF) and western blotting (WB): rat monoclonal anti-importin-α1 (1:100 for IF, 1:1000 for WB; 2G7; ab22534; Abcam), rat polyclonal anti-importin-α1 (1:100 for IF, 1:1000 for WB; I9658; Sigma-Aldrich), mouse monoclonal anti-importin-α (1:100 for IF, 1:1000 for WB; I1784; Sigma-Aldrich), mouse monoclonal anti-importin-β (1:100 for IF, 1:1000 for WB; 3E9; ab2811; Abcam), rabbit polyclonal anti-importin-β (raised against GST-tagged full-length importin-β and used at 1:1000 for WB), anti-histone H3 phospho-S10 (1:2000 for WB; 05-1336; Sigma-Aldrich), anti-α-tubulin (1:100 for IF, 1:2000 for WB; T9026; Sigma-Aldrich), anti-GFP (1:2000 for WB; sc-9996; Santa Cruz Biotechnology), anti-KIFC1 (in-house polyclonal, raised against his-tagged 887-end of KIFC1 and used at 1:100 for IF, 1:1000 for WB), rabbit polyclonal anti-TPX2 (1:100 for IF, 1:1000 for WB; 12245; Cell Signaling Technology), anti-NuMA (1:1000 for WB; ab109262; Abcam), anti-Ran (1:5000 for WB; 610340; BD Biosciences) and anti-Myc (1:1000 for WB; MABE282; Sigma-Aldrich). Kinase inhibitors were used at the following concentrations: 10 µM purvalanol A(Sigma-Aldrich), 9 µM Ro-3306 (Sigma-Aldrich), 0.1 µM BI2536 (Axon Medchem), 2 µM ZM447439(Selleck), 0.5 μM LY294009 (Selleck).
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