| 名稱(chēng) | U0126-EtOH |
| 描述 | U0126-etoh (U0126 Ethanol) is a non-ATP competitive inhibitor of MEK1 (IC50=72 nM) and MEK2 (IC50=58 nM) with selectivity. U0126-EtOH inhibited autophagy and mitophagy. |
| 細(xì)胞實(shí)驗(yàn) | HEK293 cells were maintained in Dulbecco's modification of Eagle's medium (low glucose) plus 10% foetal bovine serum. HeLa cells stably expressing wild type or kinase-dead LKB1 have been described. AMPK activity was determined by immunoprecipitate kinase assays using anti-AMPK-a1 and -a2 antibodies. Antibodies recognising AMPK phosphorylated on Thr-172 (anti-pT172), AMPK-α1 and -α2 and acetyl-CoA carboxylase-1 (ACC1) phosphorylated on Ser-80 [16] were described previously. Quantification of ratios of signals from phosphorylated and total protein using these antibodies was performed by dual labelling using the LI-COR Odyssey IR imager as described. Contents of ATP and ADP were determined for cells in 6 cm culture dishes by quickly pouring off the medium, adding 350 μl of ice-cold 5% perchloric acid, scraping the cells off with a plastic scraper, and centrifuging (14 000 · g; 3 min, 4 °C) to remove insoluble material. The perchloric acid was then extracted from the supernatant and nucleotides analysed by capillary electrophoresis of perchloric acid extracts as described previously. All incubations of cells were performed in triplicate and results are expressed as means ± S.E.M [3]. |
| 激酶實(shí)驗(yàn) | The amount of immunoprecipitated wild type MEK used in these assays was adjusted to give a similar amount of activity units as obtained with 10 nM recombinant MEK. All other assays were performed with a recombinant, constitutively activated mutant MEK-1 (ΔN3-S218E/S222D) or constitutively active MEK-2(S222E/S226D). Reaction velocities were measured using a 96-well nitrocellulose filter apparatus as described below. Unless otherwise noted, reactions were 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 were initiated by the addition of [γ-33P]ATP into the premixed MEK/ERK/inhibitor reaction mixture, and an aliquot of 100 μl was taken every 6 min and transferred to the 96-well nitrocellulose membrane plate which had 50 mM EDTA to stop the reaction. The membrane plate was drawn and washed 4 times with buffer under vacuum. Wells were then filled with 30 μl of Microscint-20 scintillation fluid, and the radioactivity of33P-phosphorylated ERK was counted with a Top Count scintillation counter. Velocities were obtained from the slopes of radioactivity versus time plots. Concentrations of ERK and ATP were 400 nM and 40 μM, respectively, unless otherwise indicated [2]. |
| 動(dòng)物實(shí)驗(yàn) | Prior to injection, FI cells were labeled with a stable fluorescent dye molecule, DiA at 10 μg/ml for 5 h at 37 1C. After washing to remove free DiA, cells were trypsinized for inoculation (U0126 experiments) or transfection (RNAi experiments). Biliary epithelial cells were injected subcutaneously, at the indicated times, into the tibia of nude mice. In the chemical experiments, 3h after inoculation, mice were treated with U0126 (10.5 mg/kg) daily by intraperitoneal injection. The length and width of each tumor were measured every day by using a caliper. The following formula was used to calculate tumor volumes ? width2 length/2. Mice were killed at the end of experiment. Tumors were immediately frozen in liquid nitrogen [5]. |
| 體外活性 | 方法：COS-7細(xì)胞用U0126-EtOH處理后檢測(cè)AP-1轉(zhuǎn)錄活性。
結(jié)果：U0126-EtOH抑制AP-1轉(zhuǎn)錄活性（IC50=1 μM）。[1]
方法：HCT116細(xì)胞用U0126-EtOH處理后使用軟瓊脂生長(zhǎng)實(shí)驗(yàn)檢測(cè)克隆形成，HeLa細(xì)胞用U0126-EtOH處理后檢測(cè)Elk1-熒光素酶報(bào)告基因。
結(jié)果：U0126-EtOH抑制貼壁非依賴(lài)性集落形成（IC50=19.4 μM），U0126-EtOH抑制EGF刺激的Elk1熒光素酶報(bào)告基因（IC50=0.29 μM）。[2]
方法：小鼠RAS-3T3細(xì)胞用U0126-EtOH處理后使用ELISA方法檢測(cè)ERK1/2磷酸化水平。
結(jié)果：10-40 μM的U0126-EtOH抑制MEK介導(dǎo)的ERK1/2磷酸化。[3] |
| 體內(nèi)活性 | 方法：為研究U0126-EtOH的抗腫瘤活性，每天將U0126-EtOH（10.5 mg/kg）腹腔注射給小鼠治療。
結(jié)果：U0126-EtOH導(dǎo)致腫瘤植入和早期生長(zhǎng)顯著減少，注射9天后的腫瘤體積減少了60-70%，此后一直保持這種狀態(tài)。[4]
方法：為研究U0126-EtOH對(duì)血管收縮的影響，大鼠接受120分鐘的暫時(shí)性中腦動(dòng)脈阻塞（tMCAO），再將U0126-EtOH（30 mg/kg）腹腔注射給大鼠。
結(jié)果：用U0126-EtOH處理后，對(duì)S6c的血管收縮顯著減少。[5] |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year
Shipping with blue ice/Shipping at ambient temperature. |
| 溶解度 | 10% DMSO+40% PEG300+5% Tween 80+45% Saline : 7.9 mg/mL (18.52 mM), Solution.
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
DMSO : 255 mg/mL (597.75 mM), Sonication is recommended.
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| 關(guān)鍵字 | virus | U0126-EtOH | U-0126-EtOH | U0126EtOH | U0126 EtOH | U-0126 | U 0126 | progeny | non-ATP | Mitophagy | Mitogen-activated protein kinase kinase | Mitochondrial Autophagy | MEK2 | MEK1 | MEK | MAPKK | MAP2K | Inhibitor | inhibit | InfluenzaVirus | Influenza Virus | competitive | Autophagy |
| 相關(guān)產(chǎn)品 | Guanidine hydrochloride | Naringin | Aceglutamide | Alginic acid | Hemin | Hydroxychloroquine | Sildenafil citrate | Stavudine | Tamoxifen | Adenosine | Paeonol | Sodium 4-phenylbutyrate |
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