Proteasome activity assay

The enzymatic activities of the proteasome were assayed using the Proteasome-Glo Cell-Based Assay Kit (Promega). The Proteasome-Glo Cell-Based Reagents each contain a specific luminogenic proteasome substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. These peptide substrates are Suc-LLVY-aminoluciferin (succinyl-leucine-leucine-valine-tyrosine-aminoluciferin), Z-LRR-aminoluciferin (Z-leucine-arginine-arginine-aminoluciferin), and Z-nLPnLD-aminoluciferin (Z-norleucine-proline-norleucine-aspartate-aminoluciferin) for chymotrypsin-like, trypsin-like, and caspase-like activities, respectively. The trypsin-like assay also contains two inhibitors to reduce nonspecific protease activities. Briefly, human CCA cells (5000 cells per well) were plated at 100 μl per well in a 96-well plate. The cells were then equilibrated at 37°C and 5% CO₂ for 12 hours and subjected to different treatments. Then, the Proteasome-Glo Cell-Based Reagents were each prepared and equilibrated at 22°C for 30 min before use. During this time, the assay plate was also equilibrated. Fifteen minutes after adding the reagent, luminescence was detected as the relative light units using the Synergy 2 Multi-Mode Reader (BioTek). The data were normalized to the cell number using the CellTiter-Glo Luminescent Cell Viability Assay (Promega).