An effective method studies the β5 subunit activity of immunoproteasome in vitro

The immunoproteasome has been found to connect multiple diseases, such as multiple myeloma and autoimmune disorders, and these antagonists of immunoproteasomes urgently need to be developed into drugs to relieve the suffering of patients. We summarized previous experiences and finally provided a simple and sensitive approach to detect β5i (the β5 subunit of the immunoproteasome) activity for research or industrial purposes. The results show that the published IC50 of ONX-0914 or bortezomib is repeatable with our assay. We also detected β5i activity in different cell lines, and the IC50 is acceptable and stable to be verified by many tests. It is worth noting that the combination of digitonin and a lower concentration of NP-40 will maintain higher immunoproteasome activity and completely lyse the cell membrane. Meanwhile, a lower concentration of DMSO is required to improve the kinetic process and reach the maximum rate of reaction. Compared to the β5c assay, the β5i commercial kit is limited to being selected. We prefer to make a highly efficient kit for the β5i assay to meet the academic or clinical needs of researchers, and it could be extensively used due to the cheaper cost and stability in the application.