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. 2009 Feb 25;83(9):4652–4669. doi: 10.1128/JVI.02408-08

FIG. 8.

FIG. 8.

EBNA3C deubiquitinates itself in a cell-free system as well as in vivo. DUBs (Myc-tagged proteins as indicated) and the ubiquitinated substrate (Flag-tagged EBNA3C 1 to 365 plus HA-tagged Ub) were expressed separately in HEK 293T cells. After immunoprecipitation with appropriate antibodies, products were washed and mixed together. The deubiquitination reaction was carried out in 100 μl of deubiquitination buffer (see Materials and Methods) at 37°C overnight (O/N). The same blots were reprobed with M2 and 9E10 for Flag-tagged and Myc-tagged proteins, respectively. The asterisks indicate the immunoglobulin bands, and arrowheads show the corresponding bands of Flag-tagged EBNA3C 1 to 365. (B) The amount of expressed Myc-tagged proteins was determined by immunoblotting with 9E10. (C) HEK 293T cells were transfected with expression vectors for HA-tagged Ub, Flag-tagged EBNA3C 1 to 365, Myc-tagged full-length EBNA3C and its various truncations as indicated, Myc-tagged EBNA1, and Myc-tagged HAUSP. Flag-tagged EBNA3C 1 to 365 proteins were IP with the M2 antibody from lysates of transfected cells prepared 36 h after transfection and analyzed by immunoblotting with HA-specific antibody (12CA5). Five percent of the whole-cell extracts of the transfected cells were subjected to SDS-PAGE and immunoblotting with 12CA5 to determine the overall extent of ubiquitination. The same blots were stripped and reprobed with M2 and 9E10 for Flag-tagged and Myc-tagged proteins, respectively. Asterisks indicate the immunoglobulin bands. IP proteins in whole-cell extracts are shown by arrowheads. (D) Ubiquitinated Flag-tagged EBNA3C 1 to 365 protein was IP similarly to what was described for panel C in the presence of either vector control (lanes 2), full-length EBNA3C (lanes 3), or catalytic mutant of EBNA3C (C143N; lanes 4) and probed for appropriate antibodies as indicated. All panels are representative pictures from similar repeated experiments. Numbers at left of blots or gels are molecular masses in kilodaltons transfected cells prepared 36 h after transfection and analyzed by immunoblotting with HA-specific antibody (12CA5). Five percent of the whole-cell extracts of the transfected cells were subjected to SDS-PAGE and immunoblotting with 12CA5 to determine the overall extent of ubiquitination. The same blots were stripped and reprobed with M2 and 9E10 for Flag-tagged and Myc-tagged proteins, respectively. Asterisks indicate the immunoglobulin bands. IP proteins in whole-cell extracts are shown by arrowheads. (D) Ubiquitinated Flag-tagged EBNA3C 1 to 365 protein was IP similarly to what was described for panel C in the presence of either vector control (lanes 2), full-length EBNA3C (lanes 3), or catalytic mutant of EBNA3C (C143N; lanes 4) and probed for appropriate antibodies as indicated. All panels are representative pictures from similar repeated experiments. Numbers at left of blots or gels are molecular masses in kilodaltons.