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GST-MEK，0.5mg GST-MAPK和40mg MBP。体积为100毫升。 20分钟后，通过加入三氯乙酸终止反应，并通过GF \u002F C过滤垫过滤。保留在过滤垫上的32P使用1205 Betaplate测定。在各种剂量范围评估PD0325901以确定剂量反应曲线。",{"key":259,"value":260},"细胞实验","将PTC细胞（1×104）接种在含有1mL培养基的24孔板中，在37℃培养箱中培养4天。在第0天将不同浓度的MEK抑制剂PD0325901一式三份加入细胞中。在第2天将溶于0.8％NaCl溶液中的5mg \u002F mL MTT加入到每个孔（0.2mL）中以测试GC50或每天用于细胞生长曲线。用MTT将细胞在37℃温育3小时。然后从孔中吸出液体并丢弃。将染色的细胞溶解在0.5mL的DMSO中，并使用Synergy HT多重检测酶标仪测量它们在570nm的吸收。对于GC50，细胞生长计算为100×（T-T0）\u002F（C-T0），其中T是48小时后用抑制剂处理的孔的光密度，T0是0时的光密度， C是仅用DMSO控制光密度。",{"key":262,"value":263},"动物实验","用鸡尾酒（100μL\u002F 10g体重的10mg \u002F mL酮 - 矿和1mg \u002F mL甲苯噻嗪）对小鼠（每组10-14只）进行麻醉。将用表达荧光素酶的逆转录病毒（5μLRPMI1640培养基中的5×10 5个细胞）稳定感染的K2和TPC-1细胞接种到甲状腺中，并使用Living Image 3.0软件每周监测小鼠的Xenogen肿瘤生长。接种一周后，通过超声处理将PD0325901溶解于80mM柠檬酸缓冲液（pH7）中，并通过口服强饲法（20-25mg \u002F kg）每天给予小鼠3周（连续5天\u002F周）。仅因肿瘤负荷或体重减轻20％而处死小鼠。用卡尺测量肿瘤大小，并通过公式（V =长度×宽度×深度）计算肿瘤体积（V）。对照小鼠单独给予80mM柠檬酸缓冲液（pH 7）。所有体内实验至少进行两次。",{"key":265,"value":266},"参考文献","[1].\nBarrett SD, et al. 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