FIG 7.

EBNA3C facilitates H2AX degradation through the ubiquitin-mediated proteasomal pathway. (A) HEK293 cells were transfected with plasmids and treated with either MG132 (20 μg/ml) or DMSO as a control for 30 h posttransfection to inhibit proteasome activity. (B) HEK293 cells were transfected with plasmids expressing EBNA3C-Flag or Myc-H2AX. (C) HEK293 cells were transfected with various combinations of expression vectors, as shown. (D) In vivo ubiquitination assays were carried out by using 50 million BJAB (EBNA3C-negative), BJAB#7, and BJAB#10 (EBNA3C-positive) cells. H2AX antibody (2 μg) was used for pulldown experiments and subsequently detected by using ubiquitin- and H2AX-specific antibodies. The input was also analyzed as a loading control by using an anti-GAPDH antibody.