Proteasome glotm assay

Proteasome activity was determined using the Proteasome Glo^TM Assay (Promega), and the assay was performed according to the manufacturer’s instructions. Briefly, site-specific luminogenic substrates were added to determine the activity of the different catalytically active sites (Suc-LLVY-aminoluciferin). Proteasome activity was determined at baseline and after a 2-h incubation period (at 37 °C, 5% CO₂) with 12.5 nM of bortezomib. The read-out was performed on a Wallac Victor plate reader.

INS-1E, A-20 and Jurkat cells, human and mouse islets were plated in duplicates or triplicates in 96-well plates and treated with 50 nM ONX-0914, a selective inhibitor of theβ5i subunit activity (Selleck Chemicals, Rungsted, Denmark, IC50: ~10 nM for β5i, [35 (link)]) or 2 μM MG132, a broad proteasome inhibitor (Sigma-Aldrich, Søborg, Denmark) or control medium for 2 hours prior to experiments. Chymotrypsin-, trypsin- and caspase-like activity was measured through luminescent assay using commercially available Proteasome-Glo^TM Assay (Promega, Nacka, Sweden) according to the manufacturer’s protocol. Depicted data are averages of either technical duplicates or triplicates as indicated. The added trypsin-like, chymotrypsin-like and caspase-like activity is referred to as total proteasome activity.