Proteasome glotm chymotrypsin like cell based assays kit

A Proteasome-GloTM Chymotrypsin-like Cell-Based Assays kit (Promega, Madison, WI, USA) was used on intact myotubes attached to culture plates according to the manufacturer’s instructions with slight modifications. Myoblasts were seeded at 10,000 cells per well in 100 μl and differentiated in 96-well plates with clear optical bottoms. After 48-h DEX and/or r-irisin treatment of differentiated myotubes, an equal volume of luminogenic substrate specified for chymotrypsin-like protease activity was added to samples. After shaking at 700 rpm using a plate shaker for 2 min and incubation at room temperature for 10 min, luminescence was detected by a luminometer (BioTek Instruments, VT, USA). To confirm assay specificity, the same number of samples was pretreated for 1 h with 10 μM epoxomicin, a proteasome inhibitor. For each sample, proteasome activity was normalized with epoxomicin-pretreated luminescence as the background signal.

A Proteasome-GloTM Chymotrypsin-like Cell-Based Assays kit (Promega, Madison, WI) was used according to the manufacturer’s instructions. Briefly, TSPCs were seeded at 10,000 cells per well with 100μl in 96-well plates. After irisin treatment, an equal volume of luminogenic substrate specified for chymotrypsin-like protease activity was added to samples. After shaking for 2min and incubation at room temperature for 10min, luminescence was detected by a luminometer (BioTek Instruments, Winooski, VT). To confirm assay specificity, the same number of samples was pretreated for 1h with 10μM epoxomicin, a proteasome inhibitor. For each sample, proteasome activity was normalized with epoxomicin-pretreated luminescence as the background signal.