Jarvius Malin, Paulsson Janna, Weibrecht Irene, Leuchowius Karl-Johan, Andersson Ann-Catrin, Wählby Carolina, Gullberg Mats, Botling Johan, Sjöblom Tobias, Markova Boyka, Ostman Arne, Landegren Ulf, Söderberg Ola
Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185, Uppsala, Sweden.
Mol Cell Proteomics. 2007 Sep;6(9):1500-9. doi: 10.1074/mcp.M700166-MCP200. Epub 2007 Jun 12.
Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.
需要改进的方法用于原位表征细胞系和组织中的翻译后修饰。例如，期望监测用抑制剂处理的人类肿瘤样本中单个受体酪氨酸激酶的磷酸化状态，以评估治疗反应。不幸的是，用于原位观察组织翻译后修饰动态的主要方法，即免疫组织化学和免疫荧光，灵敏度和选择性有限。邻近连接分析是一种新方法，通过双重识别要求提高了选择性，并通过将DNA扩增作为靶分子检测的一个组成部分提高了灵敏度。因此，我们在此建立了一种通用的原位邻近连接分析方法，以研究血小板衍生生长因子BB刺激的细胞中血小板衍生生长因子受体β（PDGFRβ）的磷酸化。用连接有DNA链修饰的针对不同物种免疫球蛋白的特异性抗体作为二级邻近探针，与来自相应物种的一对一级抗体一起使用。一级抗体与二级邻近探针结合对受体和磷酸化位点的双重识别用于生成环状DNA链；随后通过滚环扩增复制DNA环进行信号放大。我们在稳定过表达人流感血凝素标记的人PDGFRβ的人胚肾细胞中检测到酪氨酸磷酸化的PDGFRβ，这些细胞转染了β受体的猪主动脉内皮细胞，但在转染α受体的细胞中未检测到，并且在永生化的人包皮成纤维细胞BJ hTert中也检测到内源性表达的PDGFRβ。我们还在新鲜冷冻的正在愈合伤口的人瘢痕组织切片中可视化了酪氨酸磷酸化的PDGFRβ。该方法对于研究信号转导、筛选药物作用以及提高组织病理学中的诊断潜力具有重要价值。
