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. 2008 Oct 15;82(24):12009–12019. doi: 10.1128/JVI.01680-08

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Copyright © 2008, American Society for Microbiology

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FIG. 7. — Brd4 silencing does not noticeably affect EBNA1 attachment to mitotic chromosomes. HeLa cells expressing EBNA1 were transfected with siRNA against GFP (negative control) or esiRNA against Brd4. (A) Negative-control cells were blocked in mitosis, and whole-cell lysates (W) were generated and separated into soluble (S) and chromosomal pellet (P) fractions by centrifugation. Equal cell equivalents of each fraction were then analyzed by Western blotting using Brd4 or EBNA1 antibody. (B) Downregulation of Brd4 by esiRNA was confirmed by Western blotting of whole-cell lysates, compared to siRNA treatment against GFP. (C) Cells from panel B were blocked in mitosis, and whole-cell lysates (W) were separated into soluble (S) and chromosomal pellet (P) fractions. Equal cell equivalents of each fraction were then analyzed by Western blotting using EBNA1 antibody. The same blot was also probed for NAP1, a protein known to be in the soluble fraction in mitosis, as a control for proper fractionation of soluble proteins.