Figure 9.

DHX9 is recruited to the NF-κB p65–dependent promoters during viral infection. HeLa cells were either mock-infected or infected with MHV-68 at MOI of 2 for 12 h. (A and B) Cell lysates were prepared and analyzed by ChIP assays with an anti-p65, anti-RNAPII, or anti-DHX9 antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of precipitated DNA were measured by quantitative PCR (qPCR) involving primers flanking the promoter regions of IL6 (A) and IFN-β (B). The data represent mean ± SD of triplicate assays. (C) The cell lysates were prepared for ChIP, first subjected to immunoprecipitation with the anti-p65 antibody and then subjected to re-ChIP with anti-RNAPII and anti-DHX9 antibodies. Genomic DNA samples sequentially enriched in the NF-κB target gene promoters (IL6 and IFN-β promoters) were amplified by PCR and analyzed by agarose gel electrophoresis (lanes 3–6). Reciprocal re-ChIP using the anti-RNAPII or anti-DHX9 antibody was also conducted with the first antibody followed by incubation with the second antibody as indicated (lanes 7–14). Normal IgG was included as a control for nonspecific immunoprecipitation and input lanes indicate the chromatin complexes prior to immunoprecipitation. A set of representative agarose gel images is shown from two independent experiments.