Detection of Janus-activated kinase-1 and its interacting proteins by the method of luminescent oxygen channeling

Abstract

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.