Using LCA to measure real-time interaction between FGF14 and Nav1.6 C-tail in live cells.

<p>(<b>A</b>) Using the bioluminescence-based luciferase complementation assay (LCA) to measure protein:protein interactions. Two proteins of interest (FGF14, and CD4-Nav1.6-Ctail in this example) are fused to Cluc and Nluc fragments of <i>Photinus</i> luciferase. Upon interaction of the protein components, Nluc and Cluc fragments reconstitute into functional luciferase enzyme, which produces luminescence in the presence of luciferin substrate. The intensity of luminescence is linear to the strength of the protein:protein interaction identified. <b>(B)</b> Schematic of constructs used for LCA experiments, to scale. <b>Top</b>: Cluc (AAs 398–550), linker (GGGSSGGGQISYASRG), FGF14-b (AAs 1–252). <b>Bottom:</b> CD4-ΔCtail (AAs 1–395), Nav1.6-Ctail (AAs 1763–1976), linker (QISYASRGGGSSGGG), Nluc (AAs 2–416). <b>(C)</b> Schematic of protein inhibition by kinase inhibitors. ATP-competitive kinase inhibitors block the ATP-binding site of the target kinase, preventing the transfer of phosphate groups to the substrate. Non-ATP competitive kinase inhibitors work through other mechanisms, such as changing the conformation of the ATP-binding site to prevent docking of ATP.