Proteasome Activity Assay Protocol

We analyzed the proteasome activities in 25–50 μg protein extracts. The assay buffer consists of 50 mM Tris (pH 7.5), 2.5 mM EGTA, 20% glycerol, 1 mM DTT, 0.05% NP-40, and 25 μM substrate. Substrates Ac-nLPnLD-AMC, Bz-VGR-AMC and Suc-LLVY-AMC were from Enzo life science [43 (link), 68 (link)]. MG132 was used at a final concentration of 10 μM to block proteasome activities as negative controls. Fluorescence was read at 5 min intervals for 2 h, at an excitation wavelength of 380 nm and an emission wavelength of 460 nM, n ≥ 3 per group. Data were activities normalized to total protein and control in the assay.