FIG. 7.

EBNA3C can function as a DUB. (A) Alignment of EBNA3C fragments (lower row) with the consensus sequences of Ub protease (UBP) family proteins (upper row) using the T-Coffee method (55). Highlighted residues indicate cysteine (C) and histidine (H) amino acids implicated in the catalytic mechanism. (B) Myc-tagged full-length EBNA3C, EBNA1, HAUSP, or vector (pA3M) was IP with the Myc-specific antibody (9E10) from lysates of transfected HEK 293T cells and tested for the ability to cleave Ub₄. After overnight incubation at 37°C, the reaction supernatants were analyzed by 15% SDS-PAGE and silver staining (top panel). Substrate control (Ub₄) and positive control (isopeptidase T [IsopT]) are shown in lanes 5 and 6 and in lane 6, respectively. Lines indicate the positions of tetrameric (Ub₄), trimeric (Ub₃), dimeric (Ub₂), and monomeric Ub. Specific mono-Ub bands were scanned with Odyssey imager software. The amount of IP Myc-tagged proteins was determined by immunoblotting with 9E10 (bottom panel). The asterisk indicates the immunoglobulin bands. (C) Cartoon diagrams of different EBNA3C truncated fragments. Squares show the positions of consensus cysteine and histidine domains in EBNA3C. (D) Myc-tagged full-length EBNA3C or different truncated fragments of EBNA3C as indicated in panel C were subjected to an in vitro deubiquitination assay as described for panel B. The reaction supernatants were analyzed by 15% SDS-PAGE followed by silver staining (top panel). Different forms of Ub are shown by lines on the right side of the gel. The bottom panel shows the Western blot analysis of the total amount of immunoprecipitated Myc-tagged EBNA3C proteins. The asterisk indicates the immunoglobulin bands. (E) Approximately 200 μg of lysates prepared from either BJAB cells or BJAB cells stably expressing EBNA3C was incubated with HAUbVME probe (1 μg/μl) for 2 h at 37°C and subjected to immunoprecipitation with specific monoclonal antibody against HA epitope (12CA5). Samples were resolved by 7% SDS-PAGE and transferred to an 0.45-μm nitrocellulose membrane. The membrane was probed with anti-EBNA3C antibody (A10). NS, nonspecific band. (F) HEK 293T cells were transfected with either 15 μg of full-length EBNA3C-expressing construct or empty vector. Cells were harvested at 36 h posttransfection, approximately 5% of the lysed cells were saved as input, and the remainder were IP with 1.5 μg of M2 antibody against Flag epitope. IP proteins were incubated with HAUbVME probe (1 μg/μl) for 2 h at 37°C in labeling buffer (see Materials and Methods). Samples were divided into halves, resolved by 7% SDS-PAGE, and subjected to Western blotting with anti-HA antibody (top panel) and A10 for detection of EBNA3C (bottom panel). Numbers at left of blots and gels are molecular masses in kilodaltons.