Tp53bp1

Cells and tissues were solubilized in lysis buffer (NO. 20–188; Sigma-Aldrich) and the solution was boiled for 5–10 min. The protein samples were separated by SDS-PAGE, following which the proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and then incubated with primary antibodies directed against VANGL1 (dilution 1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Ki67 (dilution 1:1000, Santa Cruz Biotechnology), caspase-3 (dilution 1:1000, Santa Cruz Biotechnology), BRAF (dilution 1:500, Santa Cruz Biotechnology), TP53BP1 (dilution 1:500, Santa Cruz Biotechnology), RAD51 (dilution 1:500, Santa Cruz Biotechnology), and β-actin (dilution 1:1500, Santa Cruz Biotechnology) for 2 h at room temperature. The blots were developed using peroxidase-conjugated anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Inc.), and the proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, NJ, USA). β-actin was used as a loading control.

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.