EBNA3C is predominantly K63-linked polyubiquitinated when proteasome is inhibited.

LCLs were either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h and subjected for (A) immunostaining with EBNA3C, total ubiquitin (Ub), K48- and K63-linked polyubiqitination specific antibodies; (B) western blot analyses with indicated antibodies. (A) Nuclei were counterstained with DAPI before mounting the cells for confocal analyses. (C-D) HEK293 cells transiently transfected with flag-tagged EBNA3C expression vector, either left untreated (DMSO control) or treated with 20 μM MG132 for 4h were subjected for (C) western blot analyses or (D) immunoprecipitation (IP) under denatured conditions using anti-flag antibody followed by western blot analyses using indicated antibodies. Each panel in (A) corresponds to single experiment of two independent experiments. Scale bars, 5 μm. Western blots in (B-C) were performed by stripping and reprobing the membranes with different antibodies.