Proteasome glo assay kit

Proteasome activity was measured by employing the Proteasome-Glo™ Assay Kit for chymotrypsin-, trypsin-, and caspase-like activities, purchased from Promega (Madison, MA, USA). Briefly, for measurements of proteasome activity in cells, BT-474 cells were seeded in 96-well plates (10,000 cells/well; each condition in triplicates) in the growth medium, for 24 h. Next, cells were treated with different doses of ouabain or equal quantities of vehicle for an additional 24 h. Notably, the proteasome inhibitor Mg-132 (1 µM) was used as an internal control for all assays. After treatment, the 3 activities of the proteasome were measured according to the manufacturer’s instructions, in a Tecan-Spark Elisa reader every other 30 s, for a total period of 30 min.

The proteasomes were purified from tissue samples using Proteasome Isolation Kit (Calbiochem, Germany). Then, the proteasome activity was measured by Proteasome-Glo assay kit (Promega, WI) following the manufacturer's instruction. The total proteins from tissues were prepared and the protein concentration was determined using a BCA protein assay. The concentration of 20S proteasome, the core subunit of proteasome, was measured as described in the manufacturer's manual using 20S Proteasome ELISA Kit (Creative Diagnostics, Shirley, NY). The amount of 20S proteasome was calculated per mg total protein. Additionally, aliquots of total proteins were used for Western Blot analysis with antibodies to TP53BP1, UCHL1, UBA3, UBE2S, PSMD1 and USP12 (Santa Cruz, CA). The β-actin was used as an internal control. All experiments were independently repeated five times.