Figure 7. Real-time imaging of the dynamics of ligand-independent activation by the Ca⁺⁺ chelator EGTA.

A. EGTA treatment resulted in a linear time-dependent increase in bioluminescence from the NotchFL LCI reporter line (but not from the ligand-independent NICD LCI reporter line, right) that was inhibited by both BB94 and DAPT. B. The increase in bioluminescence (fold activation) correlated with the appearance and accumulation of the NICD cleavage product (see fig. S7 for additional Western analyses). C. Monitoring the activation process by LCI does not depend on downstream transcription and translation as it occurs in the presence of 1µg/ml ActinomycinD (ActD) or 10 µg/ml cycloheximide (CHX), demonstrating the real-time nature of the assay. D, E. Using the LCI reporter, we were able to perform detailed temporal and dose response analyses with EGTA. We demonstrate that complete activation occurs within a narrow EGTA concentration range.