Ube2s

The immunostaining assays were carried out and evaluated as described previously 12 (link). An SP-kit was used according to the manufacturer's instructions. Tissue sections were incubated with a primary antibody against Ube2S (Santa Cruz, CA, USA, dilution, 1:100).

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.