Chromatin IP assays for USP7, GMPS and Ub-H2B in EBV genomes.

<p>A. ChIP experiments were performed in Raji cells using antibodies against EBNA1 (left panel), USP7 (middle panel), GMPS (right panel) and nonspecific rabbit IgG as a negative control. Recovered DNA fragments were quantified by real-time PCR using primer sets for the <i>oriP</i> DS and FR regions or the BZLF1 promoter region. B. D98/Raji cells were treated with siRNA against USP7 or GFP (negative control), then ChIP experiments were performed as in A using antibodies against GMPS (right panel). Down-regulation of USP7 by siUSP7 treatment was confirmed by Western blotting, while GMPS levels were unaffected by this treatment (left panel). C. D98/Raji cells were treated with siRNA against USP7 or GFP and ChIP assays were performed using antibodies against histone H2B and monoubiquitylated histone H2B (Ub-H2B) and primer sets for the indicated region of the EBV genome (LMP = LMP1 promoter region). Relative ratios of Ub-H2B to total H2B were determined for each treatment and the average fold increase in Ub-H2B after siUSP7 treatment (as compared to siGFP treatment) from multiple experiments is shown.