4.11. 20S Proteasome Activity Assay

We incubated lysates (50 µg total protein/sample) at 37 °C for 60 min in the dark with the proteasome activity assay reaction buffer (50 µL/sample; 50 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic Acid (HEPES) (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 20% glycerol, 5 mM sodium pyrophosphate, 30 mM β-glycerophosphate, 30 mM sodium fluoride, and a protease inhibitor cocktail) containing 100 µM Suc-LLVY-AMC fluorogenic substrate (Enzo Life Sciences, Inc., Farmingdale, NY, USA) in 96 well plates for measuring chymotrypsin-like activity [62,66]. Fluorescence signals were measured using a 380 nm excitation filter and 460 nm emission filter to assess chymotrypsin-like activity using a SpectraMax M5 microplate reader.