DNA transfection was performed using the FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s instructions; each transfection was normalized using corresponding vector plasmids. At 24 h posttransfection, the cells were harvested and treated with TNF-α (10 ng/ml; Sigma), LPS (1 µg/ml; Sigma), or MG-132 (20 µmol/liter; Alexis). For treatment with Geldanamycin (GA), the cells were incubated with 10 µM Geldanamycin (Sigma) or with 0.1% DMSO vehicle for the indicated times. For the luciferase assay, cells were cotransfected with plasmids indicated in the figures, luciferase reporter vectors, and the Renilla luciferase reporter, pTK-Renilla-Luc (Promega, Madison, WI), which served as an internal control for transfection efficiency. Luciferase activity was measured by the Dual-luciferase assay system (Promega) according to the protocol recommended by the manufacturer. The luciferase assay was performed in triplicate, and the results are shown as means ± standard error (SE).
Geldanamycin
Manufactured by Merck Group
Sourced in United States, Germany
Geldanamycin is a natural product isolated from the bacterium Streptomyces hygroscopicus. It is a benzoquinone ansamycin antibiotic that has been studied for its potential therapeutic applications. Geldanamycin functions as an inhibitor of the heat shock protein 90 (Hsp90) chaperone, which is involved in the folding and stability of various client proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (6 mentions)
Radicicol, by Merck Group (4 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Flat bottom 96 well microtiter plates, by Corning (2 mentions)
Tert butyl peroxide, by Merck Group (2 mentions)
Geldanamycin, by AG Scientific (2 mentions)
Radicicol, by AG Scientific (2 mentions)
17 aminogeldanamycin, by BOC Sciences (2 mentions)
17 aag, by Merck Group (2 mentions)
Onalespib, by Selleck Chemicals (2 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Tnf α, by Merck Group (1 mentions)
Ptk renilla luc, by Promega (1 mentions)
Gsk690693, by MedChemExpress (1 mentions)
Evp4593, by Selleck Chemicals (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Lovastatin, by Merck Group (1 mentions)
28 protocols using geldanamycin
1
TNF-α, LPS, and MG-132 Induced Gene Expression
2
Cardioprotective Effects of Morphine and Inhibitors
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The rats were randomized into eight groups (n = 20 per group). (1) Sham group: rats underwent thoracotomy, passing the ligature through the heart but without tying it (150 min). (2) I/R group: 30 min of ischemia and followed by 2 h of reperfusion. (3) IPostC group: 30 s of ischemia altered with 30 s of reperfusion, repeated for three cycles, and followed by reperfusion for 2 h. (4) MP group: I/R followed by morphine administration (0.3 mg/kg [18 (link)] IV bolus; Sigma-Aldrich, St. Louis, MO, USA) 10 min before the beginning of reperfusion and then reperfused for 2 h. (5) MP + GA group: administration of the HSP90 inhibitor geldanamycin (GA, 1 mg/kg [19 (link)] IP; Sigma-Aldrich). (6) MP + GSK group: administration of the Akt inhibitor GSK-690693 (GSK, 20 mg/kg [20 (link)] IP; Med Chem Express, NJ, USA). (7) MP + PMX group: administration of the C5a inhibitor-PMX 205 (PMX, 1 mg/kg/day [21 (link)], administration for 28 days via drinking water; Med Chem Express, NJ, USA). (8) MP + QNZ group: administration of the NF-κB inhibitor EVP4593 (QNZ, 1 mg/kg [22 (link)]; IP, Selleck, USA). All inhibitors were administered 10 min before morphine and followed by 2 h reperfusion.
3
Glioblastoma and Pancreatic Cancer Cell Lines
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U373, T98G, and U87 (human glioblastoma cell lines with mutant and wild type p53) and Panc1 (human pancreatic cancer cell line with mutant p53) were grown in RPMI 1640 (Thermo Fisher Scientific), 10% Fetal Bovine Serum (FBS) (Corning), L- glutamine, streptomycin (100 μg/ml) (Corning), and penicillin (100 U/ml) (Corning) in 5% CO2 at 37°C. Cells were always detached using Trypsin-EDTA solution (Biological Industries, Cromwell, CT, USA).
U373, T98G, U87, and Panc1 cells were treated with AG490 (100 μM) (Millipore) for 48 h. U373 cells were treated with lovastatin (50 μM) (Sigma Aldrich) for 24 h. U373 cells were pre-treated with bortezomib (5 nM) (Santa Cruz Biotechnology) for 30 min and then treated with AG490 (100 μM) (Millipore) for 48 h. U373 and Panc1 cells were treated with geldanamycin (100 nM) (Sigma Aldrich) for 24 h.
U373, T98G, U87, and Panc1 cells were treated with AG490 (100 μM) (Millipore) for 48 h. U373 cells were treated with lovastatin (50 μM) (Sigma Aldrich) for 24 h. U373 cells were pre-treated with bortezomib (5 nM) (Santa Cruz Biotechnology) for 30 min and then treated with AG490 (100 μM) (Millipore) for 48 h. U373 and Panc1 cells were treated with geldanamycin (100 nM) (Sigma Aldrich) for 24 h.
4
Chloroplast Transporter Activity Assay
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Intact chloroplasts from 10 g of 12-day-old PsbO11–85GFP seedlings were isolated and resuspended in HMS buffer containing 10 μM chymostatin (BioShop) to inhibit serine proteases. Chloroplasts (0.2 mg ml–1 chlorophyll) were incubated at 25 °C under 120 μmol m−2 s−1 white light or in the dark (wrapped by aluminum foil) for 12–60 min. Transport in the dark was induced by adding 5 mM Mg-ATP (BioShop) to intact chloroplasts. Reactions were stopped using HMS buffer containing EDTA to a final concentration of 10 mM. Alternatively, transport assays were conducted in the presence of 10 mM sodium azide, 10 μM geldanamycin (Sigma, G3381), 10 μM 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU; Sigma-Aldrich), or 12 μM 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB; Sigma-Aldrich). DCMU and DBMIB were prepared as 1000-fold concentrated stocks in 95% ethanol prior to dilution in H2O just prior to use.
5
Dissolving Nutlin-3 and Geldanamycin
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Nutlin-3 (Cayman Chemical Company, Michigan, USA) and geldanamycin (Sigma-Aldrich, Inc., St Louis, MO, USA) were dissolved in DMSO, and stored at −80°C. When used in cell culture work, the final concentration of DMSO did not exceed 0.1%.
6
Immunoblotting and Microscopy Protocols
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Antibodies used for immunoblotting: rabbit anti-ErbB2 (29D8, Cell Signaling), mouse anti-ErbB2 (Ab-17, Thermo Fisher Scientific), rabbit anti-ErbB3 (Ab-1328, Sigma Aldrich, MO), rabbit anti-Ebp50 (SLC9A3R1, Sigma Aldrich), rabbit anti-Ebp50 (A310, Cell Signaling), mouse anti-ezrin (E8897, Sigma Aldrich), rabbit anti-radixin (HPA000763, Sigma Aldrich), rabbit anti-pERM (3149, Cell Signaling), mouse anti-GAPDH (Ab-9484, Abcam), mouse anti-pErk1/2 (9106, Cell Signaling), rabbit anti-pAkt (4058, Cell Signaling), mouse anti-Ubiquitin (P4G7, Covance). Antibodies used for microscopical analysis: rabbit anti-ErbB2 (29D8, Cell Signaling), mouse anti-ErbB2 (9G6, Santa Cruz), rabbit anti-pERM (3149, Cell Signaling), rabbit anti-ezrin (3145, Cell Signaling), mouse anti-ezrin (E8897, Sigma Aldrich), rabbit anti-radixin (HPA000763, Sigma Aldrich), mouse anti-radixin (1E12, Novus Biologicals), rabbit anti-Ebp50 (SLC9A3R1, Sigma Aldrich), mouse anti-Hsp90 (sc-13119, Santa Cruz), mouse anti-c-Cbl (Upstate). HEPES, bovine serum albumin, geldanamycin, lactacystin, chloroquine and n-octylglucopyranoside were purchased from Sigma-Aldrich. The small molecule inhibitor NSC668394 was purchased from Calbiochem, and the dynamin-inhibitor Dyngo4a was purchased from Fisher Scientific. The plasmids encoding GFP-ErbB2 and CFP-ErbB3 were kind gifts from Dr. B. van Deurs (University of Copenhagen, Denmark).
7
Investigating Receptor Signaling Pathways
8
Hypocotyl Length Assay in Arabidopsis
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bes1-2 (Lachowiec et al., 2013 (link)), bzr1-2 (GABI_857E04), beh3-1 (SALK_017577), and beh4-1 (SAIL_750_F08) are in the Col-0 background. beh1-1 (SAIL_40_D04) and beh2-1 (SAIL_76_B06) are in the Col-3 background.
For hypocotyl length assays, seeds were sterilized for 10 min in 70% ethanol, 0.01% Triton X-100, followed by 5 min of 95% ethanol. After sterilization, seeds were suspended in 0.1% agarose and spotted on plates containing 0.5× Murashige Minimal Organics Medium and 0.8% bactoagar. Seeds on plates were then stratified in the dark at 4°C for 3 days and then transferred to an incubator cycling between 22°C for 16 h and 20°C for 8 h to imitate long days. Plate position was changed every 24 h to minimize position effect for light grown seedlings. Racks of plates containing dark-grown seedlings were wrapped in foil. For HSP90-inhibitor assays, 1 μM geldanamycin (Sigma) was added to the medium. An equivalent volume of the solvent DMSO was used for a control.
For hypocotyl length assays, seeds were sterilized for 10 min in 70% ethanol, 0.01% Triton X-100, followed by 5 min of 95% ethanol. After sterilization, seeds were suspended in 0.1% agarose and spotted on plates containing 0.5× Murashige Minimal Organics Medium and 0.8% bactoagar. Seeds on plates were then stratified in the dark at 4°C for 3 days and then transferred to an incubator cycling between 22°C for 16 h and 20°C for 8 h to imitate long days. Plate position was changed every 24 h to minimize position effect for light grown seedlings. Racks of plates containing dark-grown seedlings were wrapped in foil. For HSP90-inhibitor assays, 1 μM geldanamycin (Sigma) was added to the medium. An equivalent volume of the solvent DMSO was used for a control.
9
Antibody Screening for Cancer Targets
10
Hsp90 and Related Protein Antibodies
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