Purified rabbit 20S proteasome (35 ng) was incubated with 40 μmol/L of fluorogenic peptide substrate Suc-LLVY-AMC (for the proteasomal chymotrypsin-like activities) in 100 μl assay buffer (20 mM Tris-HCl, pH 7.5) in the presence of shikonin at different concentrations or the solvent DMSO for 2 h at 37°C, followed by measurement of hydrolysis of the fluorogenic substrates using a Wallac Victor3™ multilabel counter with 355-nm excitation and 460-nm emission wavelengths.