| 名稱 | (E/Z)-TG003 |
| 描述 | (E/Z)-TG003 is a potent and ATP-competitive Cdc2-like kinase (Clk) inhibitor. |
| 細(xì)胞實驗 | 2 × 105 HeLa cells or 1.5 × 105 COS-7 cells resuspended in 2 mL of medium are plated on 6-well dishes, and 2 μL of 10 mM TG003 dissolved in Me2SO (final concentration at 10 μM), or 2 μL of Me2SO, is added to some wells. Cells are trypsinized, and the density is counted every 24 h for 3 days. Cells are then fixed with 1 mL of ice-cold 70% ethanol, washed with PBS, incubated in 1 ml of PBS containing 1 μg/mL DNase-free RNase A and 50 μg/mL propidium iodide for 20 min at 37 °C, and proceeded to cell cycle analysis by FACSCalibur.(Only for Reference) |
| 激酶實驗 | In Vitro Kinase Assay : Kinase activity of Clks and SRPKs is assayed in a reaction mixture, containing 200 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 1–20 μM ATP, 1 μCi of [γ-32P]ATP, 1 μg of synthetic peptide of SF2/ASF RS domain (NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH), and 0.1–1 μg of purified kinases in a final volume of 40 μL. cAMP-dependent protein kinase activity is assayed in a reaction mixture containing 80 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 10 μM ATP, 1 μCi of [γ-32P]ATP, 5 μg of histone H1, and 1 μg of catalytic subunit of rat cAMP-dependent protein kinase purified. Protein kinase C activity is assayed in a reaction mixture containing 200 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 1 mM CaCl2, 80 μg/mL phosphatidylserine, 8 μg/mL diolein, 10 μM ATP, 1 μCi of [γ-32P]ATP, 5 μg of histone H1, and 2 μL of partially purified rat protein kinase C. The final concentration of Me2SO is adjusted to 1% regardless of inhibitor concentration. The reaction mixture is incubated at 30 or 25 °C for mammalian or Xenopus recombinant proteins, respectively, for 10 min, and a half-portion is spotted on P81 phosphocellulose membrane. The kinase assay conditions, including the incubation period and concentration of kinases and substrates, are optimized to maintain the linearity during incubation. The membrane is washed with 5% phosphoric acid solution (SF2/ASF RS domain) or 5% trichloroacetic solution (histone H1) at least over 15 min. The radioactivity is measured using a liquid scintillation counter. The net radioactivity is deduced by subtracting the background count from the reaction mixture without kinase, and the data are expressed as the percentage to the control sample containing the solvent. |
| 體外活性 | 10 μM TG003能夠治療非洲爪蟾體內(nèi)過度Clk活性誘導(dǎo)的胚胎缺陷. |
| 體內(nèi)活性 | TG003通過抑制IL-1β異核RNA的剪接阻斷血小板產(chǎn)生IL-1β RNA。在3T3-L1脂肪細(xì)胞分化過程中,TG003也阻斷PKCβII的可變剪接和PPARγ1和PPARγ2的表達。TG003通過抑制Clk1/Sty介導(dǎo)的磷酸化抑制人類β珠蛋白在體外的SF2/ASF依賴性剪接。TG003抑制哺乳動物細(xì)胞中Clk1/Sty激酶活性,而對10 μM濃度的HeLa和COS-7細(xì)胞的生長沒有毒性作用。 |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 6 mg/mL (24.06 mM), Heating is recommended.
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | (E/Z) TG003 | Cyclin dependent kinase | (E/Z)-TG-003 | site | tumor | Wilm's | TG-003 | splice | inhibit | mutation | Inhibitor | TG 003 | CDK | (E/Z)-TG003 | cancer | (E/Z)TG003 | (E/Z)-TG 003 | breast |
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