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EDTA，2mM二硫苏糖醇，0.01％NP-40 [pH 7.5]）中孵育30分钟。室内温度。激酶反应在30°C，20μL（15μL酶+抑制剂，2μL1mM ATP），2μLγ32P-ATP（40μCi）和1μL重组Cdc25C（100μl）中进行20分钟。 ng）或酪蛋白（1μg）底物。通过在20μL2×Laemmli缓冲液中煮沸2分钟终止反应。通过18％SDS-PAGE分离磷酸化的底物。将凝胶干燥并暴露于X射线胶片3-10分钟。",{"key":240,"value":241},"细胞实验","细胞在补充有10％胎牛血清和1单位\u002F mL青霉素 - 链霉素溶液的DMEM或RPMI中生长。将肿瘤细胞以1×10 5个细胞\u002F mL \u002F孔的密度接种到6孔培养皿中，24小时后以各种浓度加入Rigosertib。在96小时的处理后，从重复的孔中测定细胞计数。通过台盼蓝排除法确定活细胞的总数。",{"key":243,"value":244},"动物实验","Bel-7402肿瘤模型：20只雌性无胸腺（NCR-nu \u002F nu）裸鼠皮下注射1×107个Bel-7402肿瘤细胞，10-14天后，当肿瘤体积达到200-250 mm时，小鼠分为四组，每组包含相同体积的肿瘤。溶解于PBS中的Rigosertib（ON01910,250mg \u002F kg）在隔天腹膜内单独给药或与奥沙利铂（100mg \u002F kg）联合给药。使用可追踪的数字游标卡尺每周两次进行肿瘤测量。在每次测量期间确定体重。观察动物的毒性迹象[1]。",{"key":246,"value":247},"参考文献","[1].\nGumireddy K, et al. ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent. Cancer Cell. 2005 Mar;7(3):275-86.\n[2].\nReddy MV, et al. Discovery of a clinical stage multi-kinase inhibitor sodium (E)-2-{2-methoxy-5-[(2',4',6'-trimethoxystyrylsulfonyl)methyl]phenylamino}acetate (ON 01910.Na): synthesis, structure-activity relationship, and biological activity. J Med Chem.\n[3].\nChapman CM, et al. ON 01910.Na is selectively cytotoxic for chronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K\u002FAKT inhibition and induction of oxidative stress. 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