Ub amc

Sciatic nerves were prepared in APB as described above and the lysates were clarified by centrifugation for 20 minutes at 14,000 × g at 4°C. Protein concentration was determined with Bradford assay (Life Technologies) and 2.5 ug was used per reaction. The DUB activity in total lysate was measured with 1 μM Ub-AMC (Fisher Scientific) in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP, 2.5 mM DTT, and 0.05 mg/mL BSA as described above.
HA-Ub-VS (Boston Biochem) was added to the lysate at a final concentration of 1 μM in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP, 2.5 mM DTT. The reaction took place at room temperature for 25 minutes and was stopped by the addition of 5X Laemmli Buffer. Samples were resolved by SDS-PAGE and transferred to PVDF membranes for western analysis.

The DUB activity of purified proteasomes was measured with 500 nM Ub-AMC (Fisher Scientific) in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP, 2.5 mM DTT, and 0.05 mg/mL BSA. For experiments with inhibitors, 1 mM TPEN (Sigma) and 2 μM IU1 (Selleckchem), inhibitors were added to the buffer prior to the addition of the buffer to the proteasomes and the beginning of the reaction. Fluorogenic hydrolysis was measured at excitation=380 nm and emission=460 nm at 37°C for 60 minutes in a BioTek Synergy plate reader. Rate of hydrolysis was calculated based on the slope of fluorescence increase over time during the linear phase of the reaction.
HA-Ub-VS (Boston Biochem) was added to purified proteasomes at a final concentration of 250 nM in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP, 2.5 mM DTT. The reaction took place at room temperature for 25 minutes and was stopped by the addition of 5X Laemmli Buffer (10% SDS, 600 mM DTT, 30% glycerol, 0.01% bromophenol blue, and 360 mM Tris-HCl pH 6.8). Samples were resolved by SDS-PAGE and transferred to PVDF membranes for western analysis.