Randall Learish1, Natasha Karassina1, Georgyi V. Los1, Chad Zimprich1, Mark G. McDougall2, Dieter H. Klaubert2, Marjeta Uhr1, Danette Hartzell1, Nidhi Nath1, Gediminas Vidugiris1, Robert F. Bulleit1, Keith Wood1
1Promega Corporation, Madison, WI 53711
2Promega Biosciences Inc., San Luis Obispo, CA 93401
The study of complex protein networks in living cells is facilitated by the use of technologies which offer multiple applications. Here we study a cell-based model of the NFκB signaling pathway using a reporter-fusion protein technology to both functionally label, and immobilize and capture, p65 and its binding partners. Fusion constructs were made using the HaloTag® Interchangeable Labeling Technology and expressed by transient or stable transfection in mammalian cell lines. p65 fusion protein was immobilized onto HaloLink™ agarose beads without prior purification steps. This approach provided for successful capture of the p65 fusion protein as well as co-capture of its binding partner IκB, as demonstrated by western blotting. Fusion protein was also captured using streptavidin-coated beads following labeling of the p65-fusion with a biotinylated HaloTag® ligand. The p65-fusion was visualized in the cytoplasm of living cells following transfection and labeling with fluorescent ligands. Following fluorescent labeling and treatment with TNFα in vivo, nuclear translocation of the p65-fusion construct was visualized by time lapse confocal imaging. At intervals after treatment, lysates were obtained, fractionated into nuclear and cytoplasmic pools, and probed. Western blot analysis revealed a time dependant degradation and reappearance of IκB in the cytoplasm, as well as correlative evidence for translocation of the p65-fusion protein into the nucleus. Finally, the same technology was applied to study the DNA promoter binding activity of the p65-fusion protein by cross-linking TNFα treated samples and then capturing DNA fragments using HaloLink™ resin. The covalent attachment and rapid binding rate of the HaloTag® ligands to the reporter protein has made possible the development of a large set of applications that span the areas of imaging, high content analysis and proteomics. Here the versatility of the technology permitted the generation of a broad set of data using a single fusion construct to study NFκB signaling in vitro and in vivo.