Proteasome activity assay

Cells were lysed in proteasome activity assay buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl₂, 0.5 mM EDTA, 2 mM ATP and 1 mM dithiothreitol) by passing them ten times through a 27-gauge needle attached to a 1 ml syringe. The BCA protein assay kit (Fisher Scientific, Hampton, NH) was used to determine the protein concentration. Approximately, 20 µg of total proteins from cell lysates was used for detection. The fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (40 µM) was used to measure the chymotrypsin-like activity of the proteasome. Z-Leu-Leu-Glu-AMC (40 µM) was used to measure the caspase-like activity of proteasome and the Boc-Leu-Arg-Arg-AMC (40 µM) was used to measure the trypsin-like activity of proteasome. Fluorescence intensity was measured at 380 nm excitation and 460 nm emission by using a plate reader (PerkinElmer, Waltham, MA, USA).