Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays
Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays
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DOI：
10.2174/138620708785204081
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发表时间：
2008-08-01
影响因子：
1.8
通讯作者：
Praestegaard, Morten
中科院分区：
文献类型：
--
作者：
Henriksen, Ulla;Fog, Jacob;Praestegaard, Morten
基于绿色荧光蛋白和免疫荧光易位分析的多路复用使在一次分析运行中轻松获取来自同一细胞的多个读数。免疫荧光分析监测内源性蛋白的易位、磷酸化和上调/下调。基于GFP的检测监测稳定表达的GFP融合蛋白的易位。这样的分析可以沿着(垂直)、跨(水平)和在(分支)信号通路之间被多路传输。给出了这些策略的例子：1)MK2-GFP检测监测MK2-GFP在刺激p38通路时从细胞核到细胞质的转位。通过将不同的免疫荧光分析应用于MK2检测，创建了用于p38途径激活的去卷积的多路HCA系统，包括MK2、p38、核因子kappa B和c-jun的检测读数。2)基于GFP的内化检测与免疫荧光检测相结合，建立了一种单次检测GPCR活性的方法：pCREB(cAMP感应器)、NFATc1(钙感应器)和ERK(G蛋白激活)。以AT1受体的激活为例。3)通过获取描述细胞形态的二级参数，细胞毒性读数可以链接到感兴趣的初级读数。这种方法被用来标记细胞毒性化合物并取消选择假阳性。以ATF6再分布试验为例进行了说明。这些多元化战略提供了一个独特的机会来提高HCA数据质量，并在药物发现过程中节省时间。从一次化验运行中，可以获得几个化验读数，以帮助用户了解测试化合物的作用模式。
Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/ down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NF kappa B, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca2+ sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.