Proteasome Chymotrypsin-like Activity Assay

As described in our previous study (Kitajima et al., 2014 (link)), proteasome activity was assessed using Proteasome-Glo^TM Assay kit (Promega) following the manufacturer’s instruction. The chymotrypsin-like activity assay was conducted using skeletal muscle homogenates in a total volume of 100 ml in opaque 96-well plates. For the assays, 100 mg of protein was added to assay buffer containing 20 mM Tris-HCl (pH 7.2), 0.1 mM EDTA, 5 mM ATP, 1 mM β-mercaptoethanol, 20% glycerol and 0.04% Nonidet P40. The proteasome reagent was added separately, and 30 min later, the luminescence was recorded as relative light units on a infinite F200 pro (Tecan). Each sample was measured in triplicate.