Figure 5.

Demonstration of higher light output of BRET3 over improved BRET2 system. A) Western blot showing size and near equivalent quantity of reporter proteins in established cell population expressing RLUC8, BRET1, BRET2, or BRET3. Blot is probed with Renilla monoclonal antibody and α-tubulin monoclonal antibody as loading control. B) Representative images showing total light signal after adjusted number of stable RLuc8 and BRET2 or RLuc8 and BRET3 cells was implanted at subcutaneous tissue depths as marked. Images were captured for 2 min after injection of 25 μg CLZ400 for BRET2 imaging and for 30 s after injection of CLZ for BRET3 imaging. Mouse images were scaled to a single color scale to show differences in signal outcome from each system. C) Bar graph showing quantitative signal strength as obtained from animal experiment as in B. In comparison with BRET2 signal using CLZ400, BRET3 signal using CLZ substrate shows significantly enhanced light output.