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  • JNK-IN-7
JNK-IN-7的可视化放大

JNK-IN-7

A non-selective JNK inhibitor

原价
¥2425-14650
价格
1940-11720
JNK-IN-7的二维码

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  • 货号: ajci14298
  • CAS: 1408064-71-0
  • 别名: 3-[[4-(二甲基氨基)-1-氧代-2-丁烯-1-基]氨基]-N-[4-[[4-(3-吡啶基)-2-嘧啶基]氨基]苯基]苯甲酰胺,JNK inhibitor
  • 分子式: C28H27N7O2
  • 分子量: 493.56
  • 纯度: >98%
  • 溶解度: ≥ 24.7 mg/mL in DMSO
  • 储存: Store at -20°C
  • 库存: 现货

Background

JNK-IN-7 is a selective JNK inhibitor with IC50 values of 1.54 nM, 1.99 nM, 0.75 nM to JNK1, JNK2, JNK3, respectively. It also inhibits phosphorylation of c-Jun, which is a direct substrate of JNK kinase.


JNK-IN-8, an analog of JNK-IN-7 with an extra flag methyl, dramatically improved in selectivity and eliminated binding to IRAK1, PIK3C3, PIP4K2C and PIP5K3, comparing to JNK-IN-7. JNK-IN-7 and JNK-IN-8 require Cys116 for JNK2 inhibition. JNK-IN-7 can indeed inhibit IRAK-1 dependent E3 ligase activity of pellino, which plays an role in the Toll receptor signaling pathway in cells at relative high compound concentrations (1–10 mM).


The IRAK1 inhibitor JNK-IN-7 inhibited the IL-1-stimulated activation of Pellino 1 in IL-1R cells, but not the Pam3CSK4-stimulated activation of Pellino 1 in RAW264.7 macrophages. JNK-IN-7 also suppressed the phosphorylation of c-Jun in Pam3CSK4-stimulated RAW macrophages, but in contrast with IL-1R cells it did not affect the activation of Pellino 1.

参考文献:
[1].? Zhang T, Inesta-Vaquera F, Niepel M et al. Discovery of potent and selective covalent inhibitors of JNK. Chem Biol. 2012 Jan 27;19(1):140-54.
[2].? Goh ET, Arthur JS, Cheung PC et al. Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system. Biochem J. 2012 Jan 1;441(1):339-46.

Protocol

Kinase experiment [1]:

Cell-Based assays for c-Jun phosphorylation

The cell-based kinase assays for c-Jun phosphorylation were carried out by using the LanthaScreen c-Jun (1 ~ 79) HeLa cell lines which stably expressed GFP-c-Jun 1 ~ 79 and GFP-ATF2 19 ~ 106, respectively. Phosphorylation was determined by measuring the TR-FRET between a terbium-labeled phospho-c-Jun specific antibody and GFP. The cells were plated in white tissue culture treated 384 well plates at a density of 10,000 cells per well in 32 μL assay medium (Opti-MEM, supplemented with 0.5% charcoal/dextran-treated FBS, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM HEPES [pH 7.3] and lacking phenol red). After overnight incubation, cells were pretreated for 90 mins with JNK-IN-7 (at indicated concentration) diluted in 4 μL assay buffer followed by 30 mins of stimulation with 5 ng/mL of TNF-α in 4 μL assay buffer (final assay volume was 40 μL). The medium was then removed by aspiration and the cells were lysed by adding 20 μL of lysis buffer (20 mM Tris-HCl [pH 7.6], 5 mM EDTA, 1% Nonidet P-40 substitute, 5 mM NaF, 150 mM NaCl and 1:100 protease and phosphatase inhibitor mix, P8340 and P2850, respectively). The lysis buffer included 2 nM of the terbium-labeled anti-c-Jun (pSer73) detection antibodies. After allowing the assay to equilibrate for 60 mins at room temperature, TR-FRET emission ratios were determined on a BMG Pherastar fluorescence plate reader using the following parameters: excitation at 340 nm, emission 520 and 490 nm; 100 ms lag time; 200 μs integration time; emission ratio = Em 520/Em 490. All data were analyzed and plotted using GraphPad Prism 4.

Cell experiment [2]:

Cell lines

Human IL-1R cells and RAW264.7 macrophages

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

0.1, 1 and 10 mM; 1 hr

Applications

In human IL-1R cells, JNK-IN-7 inhibited IL-1β-stimulated phosphorylation of c-Jun and the activation of Pellino 1. In Pam3CSK4-stimulated RAW macrophages, JNK-IN-7 also inhibited the phosphorylation of c-Jun.

参考文献:

[1]. Zhang T, Inesta-Vaquera F, Niepel M, et al. Discovery of potent and selective covalent inhibitors of JNK. Chem Biol, 2012, 19(1): 140-154.


[2]. Goh ET, Arthur JS, Cheung PC, et al. Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system. Biochem J, 2012, 441(1): 339-346.

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