Proteasome activity assay

Snap-frozen tissues and cultured NRCMs were homogenized on ice in cytosolic extraction buffer (50 mM Tris-HCl pH 7.5, 250 mM Sucrose, 5 mM MgCl₂, 0.5 mM EDTA, and 1 mM DTT). Protein concentrations were determined with bicinchoninic acid (BCA) reagents (Pierce) and equally concentrated in proteasome assay buffer (50 mM Tris-HCl pH 7.5, 40 mM KCl, 5 mM MgCl₂, and 1 mM DTT). Chymotrypsin-like, trypsin-like, and caspase-like activities were determined in the presence of 28 μM (chymotrypsin) or 14 μM (trypsin and caspase) ATP, utilizing the following fluorogenic substrates: Suc-LLVY-AMC (18 μM, Boston Biochem #S280), Ac-RLR-AMC (45 μM, Boston Biochem #S290), and Z-Leu-Leu-Glu-AMC (40 μM, Boston Biochem #S230), respectively. The plate was read at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a Spectramax M5 (Molecular Devices). Activity was combined, using a 50:25:25 ratio for relative contribution of chymotrypsin, trypsin, and caspase-like activities⁴⁹.