Expression of recombinant Nedd8 in HEK293 cells. (A and B) Expression of V5- or GST-tagged Nedd8 in HEK293 cells. HEK293 cells were transfected with vectors expressing V5-proNedd8 or GST-proNedd8. At 48 h post-transfection, cells were harvested. Extracts (50μg) were used for direct immunoblot analysis with the indicated antibodies (panel A). For pull-down experiments, extracts (500 μg) were used for glutathione matrix purification or immunoprecipitation by anti-V5 or anti-Flag antibodies (panel B). Tagged Nedd8 or its conjugates, separated by 4–20% SDS-PAGE, were detected by anti-GST (panel A), anti-V5 (panel A), or anti-Nedd8 antibodies (panel B). (C)The recombinant Nedd8 is conjugated with Cul-1. The precipitates generated in panel B were subjected to immunoblot analysis with anti-Cul-1 antibodies.