open access

https://hdl.handle.net/2115/93580

Development of a Cell-Based Assay Using a Split-Luciferase Reporter for Compound Screening.

英語

mall nuclear ribonucleoprotein (snRNP)

2023-07-01

Biological & pharmaceutical bulletin

46(7)

1010-1014

Recently, the finding of recurrent mutations in the spliceosome components in cancer has indicated that the spliceosome is a potential target for cancer therapy. However, the number of small molecules known to affect the cellular spliceosome is currently limited probably because of the lack of a robust cell-based approach to identify small molecules that target the spliceosome. We have previously reported the development of a genetic reporter to detect the cellular levels of small nuclear ribonucleoproteins (snRNPs), which are subunits of the spliceosome, using a split luciferase. However, the original protocol was designed for small scale experiments and was not suitable for compound screening. Here, we found that the use of cell lysis buffer used in blue native polyacrylamide gel electrophoresis (BN-PAGE) dramatically improved the sensitivity and the robustness of the assay. Improved assay conditions were used to discover a small molecule that altered the reporter activity. Our method may be used with other cellular macromolecular complexes and may assist in the discovery of small bioactive molecules.

学術雑誌論文

Version of Record

1347-5215