Figure 5. Monitoring NICD/RBPjk complex stability using LCI.

A. Stable lines expressing NΔE-NLuc and CLuc-RBPjκ were treated with cycloheximide (CHX) to block translation and the degradation of the NICD/RBPjκ complex was monitored by LCI in real time. The half-life of the luciferase complementation activity (t_1/2=180 min) was similar with the half-life of NICD determined using pulse-chase. Moreover, complementation activity was stabilized by the indicated proteasome inhibitors, consistent with previous studies demonstrating that NICD is ubiquitinated and degraded by the proteasome. B. Image and Western analyses of cells at 6 hr. Luciferase complementation (degradation and stabilization) correlated closely with NICD-NLuc protein (detected by α-V1744 antibody). These studies confirm that the NICD stability determines the NICD/RBPjk complex half-life. Importantly, because the Luc fusion does not alter the stability of NICD, LCI will us to accurately monitor the off-rates of pathway activation.