Binda Chantal, Parent Jean-Luc
Department of Medicine, Université de Sherbrooke, Sherbrooke, QC, Canada.
Methods Mol Biol. 2015;1234:53-67. doi: 10.1007/978-1-4939-1755-6_6.
Identification of G protein-coupled receptor (GPCR)-interacting proteins is an intense subject of current research. However, confirmation and characterization of identified interactions can be difficult with GPCRs, especially at the endogenous level. Here, we describe how we characterized the interaction between the prostaglandin D2 DP1 receptor and the intracellular L-type prostaglandin D synthase by in vitro pull-down assays using purified recombinant GST- and His-tagged proteins, by co-immunoprecipitation of overexpressed Flag- and HA-tagged proteins, and by co-immunoprecipitation of endogenous proteins.
鉴定G蛋白偶联受体（GPCR）相互作用蛋白是当前研究的一个热点。然而，对于GPCR，尤其是在内源水平上，已鉴定相互作用的确认和表征可能具有挑战性。在这里，我们描述了我们如何通过使用纯化的重组GST和His标签蛋白的体外下拉实验、过表达的Flag和HA标签蛋白的共免疫沉淀以及内源性蛋白的共免疫沉淀来表征前列腺素D2 DP1受体与细胞内L型前列腺素D合酶之间的相互作用。
