FIG. 7.

EBNA-3C, which binds ubiquitin-like proteins SUMO-1 and SUMO-3/hSMT3b in yeast, can interact with these proteins in vitro and in cells. (A) GST-binding assay with the indicated GST fusion protein and in vitro-transcribed-translated [³⁵S]-labeled EBNA-3C (IVT E3C). The first lane contains 10% of the amount of input EBNA-3C applied to each binding reaction, and subsequent lanes show the results of binding to the indicated GST fusion proteins. Coomassie staining confirmed that the GST control protein was used at a >5-fold excess relative to the GST fusion proteins (data not shown). (B) BJAB cells were transiently transfected with 10 μg of pSG5-EBNA-3C (EBNA3C) and/or pSG5-flagSUMO-1 (flag-SUMO-1) as indicated. Immunoprecipitations (IP) from these cell lysates were performed using rabbit antiserum reactive against EBNA-3C (R1.9), followed by immunoblotting with EBNA-3C-reactive monoclonal antibody (A10) and anti-Flag monoclonal antibodies (M2/M5). WB, Western blot.