In-gel proteasome peptidase activity assays were performed as described previously (13), with minor modification. Cultured NRVMs were lysed on ice in cytosolic extraction buffer [50 mM tris-HCl (pH 7.5), 250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, and 1 mM adenosine 5′-triphosphate (ATP)]. The cell lysates or myocardium homogenates were plunged through a 29-gauge needle eight times with an insulin syringe before centrifugation at 4°C for 30 min (15,000g). Protein concentration was determined with BCA reagents, and samples were diluted with 4× native gel loading buffer [200 mM tris-HCl (pH 6.8), 60% (v/v) glycerol, and 0.05% (w/v) bromophenol blue]. Each well of a 4.0% native polyacrylamide gel was loaded with 40 μg of sample, and the gel electrophoresis was performed at a constant current of 15 mA for 8 hours. After electrophoresis, the gel was incubated in developing buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM ATP] containing 50 μM Suc-LLVY-AMC at 37°C for 30 min and visualized in an ultraviolet trans-illuminator with a wavelength of 365 nm. Immediately after the in-gel peptidase activity assay, the proteins from the native gel were transferred to a PVDF membrane at a constant current of 250 mA for 90 min and immunodetected with anti-Psmb5 antibodies.

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