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CO 3,27.5mM NaHCO 3,50mM NaCl的50μL体积的包被缓冲液中，用2μg\u002F孔的蛋白质或肽底物包被平板。 3mM NaN3。在室温（RT）下孵育过夜后，用50μL测定缓冲液洗涤涂覆的板一次。将测试化合物和酶与33P-γ-ATP（3.3μCi\u002F nmol）组合，总体积为20μL。将反应混合物在室温下孵育2小时并通过抽吸终止。随后用0.05％Tween-PBS缓冲液（PBST）洗涤微量滴定板6次。加入闪烁液（50μL\u002F孔）并测量33P。使用MicroBeta闪烁计数器进行液体闪烁光谱测定。荧光素酶偶联化学发光测定法在384孔白色中等结合微量滴定板中进行反应。第一步，将酶和化合物合并，孵育60分钟;通过加入反应开始反应ATP和p肽底物（聚（Glu，Tyr）4：1）在20μL的最终体内，并在室温下孵育2-4小时。激酶反应后，加入20μL等份的Kinase Glo，并使用Victor平板读数器测量发光信号。 ATP总消耗量限制在50％。 AlphaScreenTM酪氨酸激酶试验使用包被有链霉抗生物素蛋白和包被有PY100抗磷酸酪氨酸抗体的受体珠的供体珠。生物素化的聚（Glu，Tyr）4：1用作底物。通过在供体 - 受体珠复合物形成后通过发光添加供体\u002F受体珠来测量底物磷酸化。将激酶和测试化合物合并并预孵育60分钟，然后在384孔白色中等结合微量滴定板中加入ATP和总体积为20μL的生物素化聚（Glu，Tyr）。将反应混合物在室温下温育1小时。通过添加10μL含有75mM Hepes，pH 7.4,300mM NaCl，120mM EDTA，0.3％BSA和0.03％Tween-20的15-30μg\u002F mL AlphaScreen珠悬浮液来淬灭反应。在室温下孵育2-16小时后，使用AlphaQuest读数器读板。",{"key":246,"value":247},"细胞实验","将B16F10，A549和HT29细胞（每孔1.2×103）与软琼脂混合，并接种在含有10％FBS和EXEL-2880的96孔板上的基础琼脂层上。对于含氧量正常的条件，将板在21％氧气，5％CO 2和74％氮气中孵育（37℃）12至14天，而在低氧条件下孵育（37℃）在缺氧室中进行1 ％氧气，5％CO2和94％氮气。在添加50％Alamar Blue和荧光检测后，在每种条件下评估菌落数。",{"key":249,"value":250},"动物实验","在幼稚小鼠或携带B16F10肿瘤的小鼠中进行体内靶调节研究。通过口服强饲法以10mL \u002F kg施用Foretinib或载体（0.9％生理盐水）。为了检查肝脏中的Met磷酸化，在收获前10分钟静脉内施用HGF（10μg\u002F小鼠）。为了检查肺中的Flk-1 \u002F KDR磷酸化，在收获前30分钟静脉注射VEGF（10μg\u002F小鼠）0.5小时后。通过免疫印迹分析确定受体磷酸化分析。",{"key":252,"value":253},"参考文献","[1].\nQian F, et al. 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