Detection of Janus-activated kinase-1 and its interacting proteins by the method of luminescent oxygen channeling
Detection of Janus-activated kinase-1 and its interacting proteins by the method of luminescent oxygen channeling
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发光氧通道法检测 Janus 激活激酶 1 及其相互作用蛋白
DOI：
10.1039/c6ra27424b
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发表时间：
2017-01-01
期刊：
影响因子：
3.9
通讯作者：
Liu，Tian-Cai
中科院分区：
文献类型：
--
作者：
Guo，Xin-Xin;Wu，Han-Tao;Liu，Tian-Cai
Janus激活激酶1（JAK 1）在JAK-STAT和SOCS等多种信号通路中发挥重要作用。JAK 1-STAT 3通路的激活通过促进炎症反应在肿瘤炎症和免疫中起关键作用。其相互作用蛋白的检测在许多疾病中具有重要意义。虽然发光氧通道通常用于免疫测定技术，但其在JAK 1和相互作用蛋白的检测中的用途迄今尚未报道。我们构建了真核表达质粒pJAK 1-His和pJAK-STAT 3-His，并在单次转染和共转染后，通过western blotting检测其表达。然后使用免疫共沉淀和发光氧通道来鉴定蛋白质相互作用。JAK 1和STAT 3蛋白被证明是成功的过表达，免疫共沉淀法确定了它们在体外的相互作用。我们还检测了JAK 1与白细胞介素4受体（IL 4 R）、JAK 1与生长激素受体（GHR）之间的蛋白质相互作用。总之，我们验证了JAK 1和STAT 3在体外相互作用，使用免疫共沉淀，并首次展示了潜在的应用发光氧通道检测JAK 1及其相互作用蛋白。此外，我们认为这种检测方法可以帮助其他研究人员找到未报道的蛋白质，这些蛋白质在体外也有相互作用。
Janus-activated kinase-1 (JAK1) plays an important role in many signaling pathways, including the JAK–STAT and SOCS pathways. The activation of the JAK1–STAT3 pathway is a key role in tumor inflammation and immunity by promoting inflammation. The detection of its interacting proteins is of great significance in many diseases. Although luminescent oxygen channeling is commonly used in immunoassay technologies, its use in the detection of JAK1 and interacting proteins has not been reported to date. We constructed eukaryotic overexpression plasmids pENTER–JAK1-His and pENTER–STAT3-His and, following single transfection and co-transfection, measured their expression by western blotting. Co-immunoprecipitation and luminescent oxygen channeling were then used to identify the protein interactions. JAK1 and STAT3 proteins were shown to be successfully overexpressed, and co-immunoprecipitation identified their interaction in vitro. We also detected protein interactions between JAK1 and interleukin 4 receptor (IL4R), JAK1 and growth hormone receptor (GHR). In conclusion, we verified that JAK1 and STAT3 interact in vitro using co-immunoprecipitation, and demonstrated for the first time the potential application of luminescent oxygen channeling for detecting JAK1 and its interacting proteins. Also we think this assay could help other researchers find unreported proteins that also have interactions in vitro.