U0126-EtOH

Catalog No.S1102

U0126-EtOH is a highly selective inhibitor of MEK1/2 with IC50 of 0.07 μM/0.06 μM in cell-free assays, 100-fold higher affinity for ΔN3-S218E/S222D MEK than PD98059.

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U0126-EtOH Chemical Structure

U0126-EtOH Chemical Structure
Molecular Weight: 426.56

Validation & Quality Control

Product Use Citation(56)

Customer Product Validation(11)

Quality Control & MSDS

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  • Inhibition Profile
  • U0126-EtOH Mechanism

Product Description

Biological Activity

Description U0126-EtOH is a highly selective inhibitor of MEK1/2 with IC50 of 0.07 μM/0.06 μM in cell-free assays, 100-fold higher affinity for ΔN3-S218E/S222D MEK than PD98059.
Targets MEK2 [1]
(Cell-free assay)
MEK1 [1]
(Cell-free assay)
PKC [6]
(Cell-free assay)
CDK2 [6]
(Cell-free assay)

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IC50 0.06 μM 0.07 μM >10 μM >10 μM
In vitro U0126-EtOH functionally antagonizes AP- 1 transcriptional activity and blocks the production of a variety of cytokines and metalloproteinases involved in the inflammatory response. [1] U0126-EtOH inhibits T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs without effect on IL-2-induced proliferation by down-regulating IL-2 mRNA levels. [2] A recent study shows that U0126-EtOH antagonizes resveratrol-induced apoptosis in castration-resistant human prostate cancer C4-2 cells, inhibits mitochondrial function and shifts cells to aerobic glycolysis independently of MEK. [3]
In vivo U0126-EtOH, as the inhibitor of intracellular Raf/MEK/ERK signaling pathway, demonstrates antiviral activity by suppressing propagation of the 2009 pandemic IV H1N1 variant and highly pathogenic avian influenza viruses (HPAIV) in vivo in the mouse lung by inhibiting. [4] U0126-EtOH shows the potential neuroprotective effect and improving spatial learning in Morris water maze (MWM) by activating peroxisome proliferator-activated receptor gamma coactivator-1a, nuclear respiratory factor 1, and mitochondrial transcription factor A in Aβ-injected rats. [5]
Features A chemically synthesized and highly selective inhibitor of both MEK1 and MEK2.

Protocol(Only for Reference)

Kinase Assay:

[1]

In Vitro Kinase Assays The amount of immunoprecipitated wild type MEK used in these assays is adjusted to give a similar amount of activity units as obtained with 10 nM recombinant MEK. Reaction velocities are measured using a 96-well nitrocellulose filter apparatus as described below. Unless otherwise noted, reactions are carried out at an enzyme concentration of 10 nM, in 20 mM Hepes, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mg/mL BSA, pH 7.4, at room temperature. Reactions are initiated by the addition of [γ-33P]ATP into the premixed MEK/ERK/inhibitor reaction mixture, and an aliquot of 100 μL is taken every 6 minutes and transferred to the 96-well nitrocellulose membrane plate which has 50 mM EDTA to stop the reaction. The membrane plate is drawn and washed 4 times with buffer under vacuum. Wells are then filled with 30 μL of Microscint-20 scintillation fluid, and the radioactivity of 33P-phosphorylated ERK is counted with a Top Count scintillation counter. Velocities are obtained from the slopes of radioactivity versus time plots. Concentrations of ERK and ATP are 400 nM and 40 μM, respectively, unless otherwise indicated. For all of the in vitro enzyme assays, the percent inhibition is calculated 100 (1 −Vi/Vo) where Vi and Vo are the initial reaction velocities in the presence and absence of inhibitor, respectively. The data are then plotted as percent inhibition as a function of inhibitor concentration and fit, by nonlinear least squares regression, to the standard equation for a Langmuir isotherm to determine the IC50. As reported, enzyme concentrations are based upon molecular weights and mass of protein used in the final assay volume and not on active site titration. Thus, the actual enzyme active site concentration may differ from that reported.

Cell Assay:

[2]

Cell lines A.E7 or Th17 cells
Concentrations 0 to 10 μM
Incubation Time 48 hours
Method

A.E7 or Th17 cells are incubated with mitomycin C-treated B10.BR or BALB/c splenocytes plus varying concentrations of pigeon cytochrome c or PR8 Ag, or with 5 U/mL human rIL-2. In addition, some assays contains U0126 or an inactive analogue, U0124, to determine direct effects of MEK inhibition on T cell proliferation. Two days after culture initiation, each well is pulsed with 1 µCi of [3H]TdR and harvested the following day. The incorporation of [3H]TdR into DNA is quantitated on a Packard Matrix 96 direct beta counter without the use of liquid scintillation mixtures.

Animal Study:

[4]

Animal Models Female C57Bl/6 mice infected by Mouse-adapted highly pathogenic avian influenza A/FPV/Bratislava/79 (H7N7; FPV) virus and swine origin human influenza A virus (SOIV) A/Regensburg/D6/2009 (H1N1v; RB1).
Formulation U0126-EtOH is dissolved in 10% DMSO, 30% of Cremophor EL and 60% PBS.
Dosages ≤10 mM
Administration Administered via aerosol.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDog
Weight (kg)0.020.151.80.40.0810
Body Surface Area (m2)0.0070.0250.150.050.020.5
Km factor36128520
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

References

[1] Duncia JV, et al. Bioorg Med Chem Lett. 1998, 8(20), 2839-2844.

[2] DeSilva DR, et al. J Immunol. 1998, 160(9), 4175-4181.

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Chemical Information

Download U0126-EtOH SDF
Molecular Weight (MW) 426.56
Formula

C18H16N6S2.C2H6O

CAS No. 1173097-76-1
Storage 3 years -20℃powder
6 months-80℃in solvent
Synonyms N/A
Solubility (25°C) * In vitro DMSO 85 mg/mL warmed (199.26 mM)
Water <1 mg/mL (<1 mM)
Ethanol <1 mg/mL (<1 mM)
In vivo 10% DMSO+50% PEG 300+ddH2O 28mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
Chemical Name 2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile,ethanol

Customer Product Validation (11)


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Rating
Source J Natl Cancer Inst 2012 104(21), 1673-9. U0126-EtOH purchased from Selleck
Method Western Blot
Cell Lines primary human melanocytes
Concentrations 1 μM
Incubation Time 18 h
Results In BRAFV600E melanoma cells, the highly selective BRAFV600E inhibitor GDC-0879 and three selective MEK inhibitors [PD184352/CI-1040, U0126, PD98059] did not suppress c-Jun levels, although they effectively reduced phospho-ERK levels.

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Source Proc Natl Acad Sci U S A 2014 111(15):E1528-37. U0126-EtOH purchased from Selleck
Method Western blot
Cell Lines J-Lat cells
Concentrations 10/20/40 uM
Incubation Time 24 h
Results J-Lat cells were stimulated with U0126 and TPA as escribed. Samples were analyzed by Western blot to detect phosphorylated MAPK p42/p44. TPA induced robust MAPK phosphorylation, which was repressed by U0126 in a dose-dependent manner.

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Source Biomaterials 2011 32, 7524-7531. U0126-EtOH purchased from Selleck
Method Cell migration tracking and quantification
Cell Lines Stromal cells
Concentrations 0-1 nM
Incubation Time
Results Partial ERK inhibition with 0.3 nM of U0126 (data not shown) significantly increased MSC persistence time by 30% from 17 min to 22 min(Fig. B) and increased MFP by 50%, from 16 m m to 24 m m (Fig. A). Speed was decreased mildly, by <10% (Fig. C)

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Source Cell Death Differ 2012 19, 1908-16. U0126-EtOH purchased from Selleck
Method Real-time RT-PCR/ Determination of apoptosis/Immunoblot analysis
Cell Lines MCF10A cells
Concentrations 10 μM
Incubation Time 15 h
Results Inhibition of the ERK1/2 pathway by the MAPK/ERK kinase 1 (MEK1) inhibitor U0126 blocked FLIPL downregulation induced by EGF re-addition to EGF-deprived cells.

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Source J Invest Dermatol 2012 132, 2780-90. U0126-EtOH purchased from Selleck
Method Western blot
Cell Lines UACC62 cells
Concentrations 1 μM
Incubation Time 6 h
Results Induced p-Erk1/2 was associated with hyperphosphorylation of its target p90 ribosomal S6 kinase 1, which could significantly be suppressed by the Mek inhibitor, U0126.

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Source J Immunol 2010 184, 7219-7228. U0126-EtOH purchased from Selleck
Method Western blotting
Cell Lines Il6ra-/-mice
Concentrations 0.4 μg/μL
Incubation Time 0-10 d
Results Stat3 phosphorylation was reduced to a similar extent in Il6-/-, Il6ra-/-, and Il6-/-/Il6ra-/-mice (Fig. A) compared with WT mice. phosphorylation of the MAPK ERK1/2 was undetectable in Il6-/-mice yet increased in Il6ra-/-relative to WT mice 1 d after wounding (Fig. A). Topical application of U0126 reduced ERK phosphorylation in wounds of both WT and Il6ra-/- mice compared with vehicle-treated controls (Fig. B). The difference in wound contraction rates was significantly delayed in Il6ra-/-mice treated with U0126 compared with vehicle control-treated mice but did not reach significance in WT mice (Fig. C).

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Source Lung Cancer 2011 73, 274-82. U0126-EtOH purchased from Selleck
Method Cell viability assay
Cell Lines A549 cells/ H322 cells
Concentrations 2.5 μM
Incubation Time 72 h
Results When 2.5 μM U0126 was added to the gemcitabine/AG1478 combination in A549 (Fig. A), reduction in cytotoxicity was observed for 0.5 μM AG1478 but this effect became less apparent with increasing AG1478 concentration. This is represented as increased CI (greater antagonism) at low concentrations of AG1478 (Fig. B). Similarly in H322 (Fig. C), addition of U0126 attenuated cytotoxicity at 0.5 μM and 2.5 μM AG1478 but this effect was lost at higher AG1478 concentrations. This was seen as reduced synergism on the CI curve (Fig. D).

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Source Biochim Biophys Acta 2013 1832(12):1998-2008. U0126-EtOH purchased from Selleck
Method Immunohistochemistry staining
Cell Lines C57BL/6 male mice
Concentrations 10 mg/kg
Incubation Time 24 h
Results In the sham-operated kidneys, Na,K-ATPase was localized basolaterally in a stripe pattern in proximal tubular epithelial cells. In the ischemic injured kidneys, Na,K-ATPase was much fainter and distributed over the entire cell membrane of proximal tubular epithelial cells, which was treated by U0126,compared with vehicle.

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Source Biochim Biophys Acta 2013 1832(12):1998-2008. U0126-EtOH purchased from Selleck
Method Immunohistochemistry
Cell Lines C57BL/6 male mice
Concentrations 10 mg/kg
Incubation Time 24 h
Results Nine days after ischemia, tubular atrophy, dilation, and congestion of tubules were significantly greater in the U0126-treated mouse kidneys than in the vehicle-treated mouse kidneys. The number of interstitial cells also was greater in the U0126-treated mouse kidneys when compared with that in the vehicle-treated mouse kidneys. Consistent with the morphological evaluation, tubular damage scores were significantly higher in the U0126-treated mouse kidneys than in the vehicle-treated mouse kidneys.

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Source 2010 Dr. Zhang of Tianjin Medical University. U0126-EtOH purchased from Selleck
Method Western blotting
Cell Lines Breast cancer cells
Concentrations 0-10 nM
Incubation Time 24 h
Results U0126 treatment resulted in a reduction of Erk phosphorylation in Breast cancer cells.

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Source U0126-EtOH purchased from Selleck
Method Western Blot
Cell Lines HeLa cells
Concentrations 0/30/300/3000 nM
Incubation Time 24 h
Results

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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