15dPGJ2-Cys was prepared in 1 ml PBS containing 0.5 mg/ml L-Cysteine (Sigma), 1 mg GSH transferase from equine liver (Sigma), and 100 μg 15dPGJ2 (Cayman Chemical) at 37°C on a thermoblock, shaking for 2 h. The addition of glutathione-S-transferase (GST) to the 15dPGJ2-L-Cysteine mixture has been described previously (28 (link)) but was found to be expandable in order to achieve full conjugation. The reaction was stopped by addition of formic acid to a final concentration of 0.3% and the sample was subsequently loaded on a polymeric reverse phase sorbent column (StrataX-C18, Phenomenex). After the sample was loaded, the cartridge was rinsed with 1 ml 0.1% formic acid/ 5% methanol and the sample was eluted with 0.1% formic acid in methanol. Eluates were dried in a vacuum concentrator and the product obtained was dissolved in 0.5 ml methanol containing 1 mg/ml CeCl3.H2O and an aqueous solution of NaBH4 (12% w/w). The reduction reaction from a carbonyl to a hydroxyl at position C11 was carried out on ice for 1 h. Subsequently, the sample was acidified and applied to a polymeric reverse phase sorbent column and cleaned as described above. The eluate was dried and stored at −20°C until LC-MS/MS analysis. Conjugation quality and efficiency was assessed by LC-MS/MS analysis and full conjugation was assumed when no nonconjugated 15dPGJ2 could be detected.
15d pgj2
Manufactured by Cayman Chemical
Sourced in United States
15d-PGJ2 is a laboratory reagent produced by Cayman Chemical. It is a naturally occurring prostaglandin metabolite that may be used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gw9662, by Cayman Chemical (6 mentions)
Biotinylated 15d pgj2, by Cayman Chemical (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
N acetylcysteine, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
T0070907, by Cayman Chemical (3 mentions)
Mk0524, by Cayman Chemical (3 mentions)
Cay10471, by Cayman Chemical (3 mentions)
Gw9662, by Merck Group (3 mentions)
Pgf2α, by Cayman Chemical (3 mentions)
Tempol, by Bio-Techne (2 mentions)
Pyrrolidine dithiocarbamate pdtc, by Merck Group (2 mentions)
N 2 mercaptopropionylglycine mpg, by Merck Group (2 mentions)
Gsh ethyl ester, by Merck Group (2 mentions)
Dh 15d pgj2, by Cayman Chemical (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Pd169316, by Merck Group (2 mentions)
Troglitazone, by Cayman Chemical (2 mentions)
Ciglitazone, by Cayman Chemical (2 mentions)
Rosiglitazone, by Cayman Chemical (2 mentions)
34 protocols using 15d pgj2
1
Synthesis of 15dPGJ2-Cysteine Conjugate
2
Inhibiting 15-PGDH Activity in Mice
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CAY10397, a selective inhibitor of 15-Pgdh, was synthesized as previously described and dissolved in 3.3% v/v cell culture grade DMSO and 0.1M sodium carbonate following dilution with PBS (17 (link)). One day prior to infection, C57BL/6 mice on Se-A diet were administered 75 mg/kg of CAY10397 via oral gavage, a concentration that was effective in inhibiting 15-Pgdh activity in tissues by ~65% (17 (link)). These mice were gavaged every alternate day until day 21 PI. To examine the role of 15d-PGJ2 in Se-D mice, 15d-PGJ2 (Cayman Chemicals) was used. 15d-PGJ2 was evaporated under a stream of nitrogen and dissolved in PBS. One day prior to infection, C57BL/6 mice on Se-D diet were treated with 0.05 mg/kg of 15d-PGJ2 via intraperitoneal injection. The mice were treated daily until they cleared the infection.
3
Compound Sources for Biological Assays
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The compounds dimethyl fumarate (DMF), trans-chalcone, curcumin, sulforaphane, tert-butylhydroquinone (tBHQ) and dimethyl itaconate (DMI) were purchased from Sigma-Aldrich. 4-OI was chemically synthetized as described elsewhere [9 (link)]. The prostaglandins 15d-PGJ2, PGE2 and PGD2 were purchased from Cayman Chemicals, while epoxycyclopentenone (EC), cyclo-epoxycyclopentenone (cEC), EC-reduced were synthetized as previously described [[31] (link), [32] (link), [33] (link)]. The antioxidant N-acetylcysteine (NAC) and the pan-caspase inhibitor Q-VD-OPh (QVD) were purchased from Sigma-Aldrich, whereas the necroptosis inhibitor Nec-1s was from Merck Millipore.
4
Preparation of 15d-PGJ2 for Cell Culture
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15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2, #18570, Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved under sterile conditions in dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA). Aliquots (20 mM) were stored at −20 °C and used within two weeks. If not otherwise stated, DMSO has been used as vehicle control at a concentration corresponding to 20 µM 15d-PGJ2 (0.04%, v/v). All cell culture media and supplements were from Gibco (Thermo Fisher Scientific, Schwerte, Germany). Fetal calf serum (FCS, Thermo Fisher Scientific) was heat-inactivated in house (56 °C for 30 min).
5
Measurement of PDI Thiol Reductase Activity
6
Antibody Preparation and Reagent Sourcing
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Antibody against FLAG was purchased from Sigma-Aldrich, GFP and HA from Santa Cruz Biotechnology. Antibody against eIF4GI was prepared in our laboratory (Kim et al., 2005 (link)). Chemicals 15d-PGJ2, biotinylated 15d-PGJ2 and PGE2 were purchased from Cayman Chemical. Sodium arsenite was purchased from Sigma-Aldrich. Immobilized streptavidin agarose was purchased from Pierce.
7
Hypoxia-Induced HUVEC Response
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Human umbilical vein endothelial cells (HUVECs; passages, 5–8; Lonza, Basel, Swiss) were cultured in Endothelial Growth Medium-2 (Lonza) containing 10% fetal bovine serum under normoxia (5% CO2 and 20% O2). HUVECs were starved for 4 h in Endothelial Basal Medium-2 (Lonza) containing 2% fetal bovine serum under normoxia and then incubated for 6 h under normoxia (5% CO2 and 20%–21% O2) or hypoxia (5% CO2 and 5% O2). In addition, an L-PGDS inhibitor (AT-56, 100 μM; Cayman chemical) and 15-deoxy-Δ12,14 PGJ2 (15d-PGJ2, 3 μM; Cayman chemical) were pretreated 5 min before the incubation.
The total RNA of HUVECs was extracted, and cDNA was obtained by reverse transcription. The cDNA was amplified by using specific primers (supplemental Table S2 ). The mRNA expression levels were quantitated with the ΔΔCt method, using 18S-rRNA levels as internal controls.
The total RNA of HUVECs was extracted, and cDNA was obtained by reverse transcription. The cDNA was amplified by using specific primers (
8
Ubiquitin Regulation in Neurodegeneration
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15dPGJ2 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibody sources were as follows: mouse monoclonal anti-mono- and poly-ubiquitinated proteins antibody (clone FK2) and anti-poly-ubiquitinated proteins antibody (clone FK1) were from Enzo Life Sciences (Plymouth Meeting, PA, USA). Monoclonal anti-ubiquitin antibody was from Covance (Berkeley, CA, USA) and anti-ubiquitin Lys48-specific antibody was from Millipore (Temecula, CA, USA), anti-ubiquitin Lys63-specific antibody was from Abcam (Cambridge, MA, USA); anti-PARP, anti-caspase-3, and anti-Neurofilament-L antibodies were from Cell Signaling (Boston, MA, USA); anti-β-actin antibody and anti-UCH-L1 antibody were from Sigma-Aldrich (St Louis, MO, USA); anti-NeuN antibody was from Millipore; and anti-GAPDH antibody was from Ambion (Grand Island, NY, USA). Cy3- and Alexafluor 488-conjugated secondary antibodies were from Jackson Immunoresearch Lab (West Grove, PA, USA). Ultra performance liquid chromatography organic solvents and water were from VWR (West Chester, PA, USA). The lentiviral expression vector, pLVX-IRES-ZsGreen1 vector, and Lenti-X HTX concentrator and packaging system were purchased from Clontech Laboratories (Mountain View, CA, USA). WST-1 cell proliferation assay kit and Lenti-X 293 T cell line were purchased from Clontech.
9
Oxidative Stress Modulation in MG-63 Cells
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MG-63 cells were treated with indicated concentrations of 15d-PGJ2 (Cayman, MI, USA) for indicated time periods. Cells were incubated for 30 min with either an inhibitor of p38 MAPK (25 μM PD169316, Merck Biosciences, Darmstadt, Germany), N-acetyl-l -cysteine (NAC, 5 mM), pyrrolidine dithiocarbamate (PDTC, 1 mM), N-2-mercaptopropionylglycine (MPG, 5 mM) (Sigma–Aldrich, MO, USA), Tempol (1 mM, Tocris, MA, USA), PPARγ antagonist (20 μM T0070907), D-type prostanoid receptor (DP1) antagonist (100 nM MK0524), DP2 antagonist (1 μM CAY10471) (Cayman), l -buthionine-(S,R)-sulfoximine (BSO, 5 μM), GSH ethyl ester (10 mM, Sigma–Aldrich), LY294002 (10 μM) or Akt inhibitor (Akt-I, 10 μM) (Calbiochem, CA, USA) prior to 15d-PGJ2 treatment. Alternatively, cells were treated with 9,10-dihydro-15d-PGJ2 (dh-15d-PGJ2, 20 μM, Cayman) for indicated time periods.
10
Lovastatin Modulates Prostaglandin Synthesis
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Cells seeded in 24-well plates at a density of 2 × 105 cells per well and grown for 24 h were preincubated with NS-398 (1 μM) or its vehicle for 1 h. Thereafter, cells were incubated with vehicle or lovastatin lactone in the presence or absence of NS-398 for another 24 h. The final volume of the supernatant was 300 μl per well. Afterwards, cell culture media were removed and analyzed for PGE2, PGD2 and 15d-PGJ2 using enzyme immunoassay kits (PGE2, PGD2: Cayman Chemical, Ann Arbor, MI, USA; 15d-PGJ2: Enzo Life Sciences). For indication of percent control PG levels were normalized to whole cell protein and subsequently expressed as percent of vehicle control (100%).
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