DDX5 localizes to promoters of DNA replication genes and functions in the loading of RNA polymerase II onto these promoters. DDX5 localizes to promoters of DNA replication genes and functions in the loading of RNA polymerase II onto these promoters. A, Western blot (WB) analysis of E2F1 and DDX5 in DDX5 and immunoglobulin G (IgG) immunoprecipitation (IP) samples from nuclear extracts prepared from HCT116 cultures (top) and analysis of the interaction of in vitro transcribed and translated radiolabeled DDX5 with GST-E2F1 purified from bacteria and immobilized on glutathione beads (bottom). B, Q-PCR analysis of DDX5 chromatin immunoprecipitation (ChIP) samples at the indicated promoters. Cells were serum-starved, restimulated with serum for 2 hours, and then harvested for ChIP. Note that the CD4 promoter is used as negative control. Error bars show the SDs for duplicate experiments. The green bars correspond to results for the DDX5 ChIPs, whereas the black bars correspond to results for the IgG control ChIPs. All primer pairs used for Q-PCR amplify within 200 bp of the transcription start sites of the indicated genes. Q-PCR analysis of E2F1 ChIP (C), acetylated histone H3 ChIP (D), RNA polymerase II ChIP (E), and TFIIB ChIP (F) at the indicated promoters in asynchronous cells transfected with either DDX5si2008 (blue bars) or EBNA1si1666 (red bars) siRNAs. Error bars show the SDs for duplicate experiments. Neither the GAPDH nor CD4 transcripts are downregulated by DDX5 knockdown and therefore their promoters are used as negative controls in for the ChIP experiments presented in (D–F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Anthony Mazurek et al. Cancer Discovery 2012;2:812-825 ©2012 by American Association for Cancer Research