Binding of IRF-8 to target gene promoters and to the IECS in vivo. (A) Binding of IRF-8 and PU.1 to endogenous target gene promoters detected by chromatin immunoprecipitation (ChIP) assay. Tot2 cells were transduced with empty MSCV or MSCV–IRF-8, and were analyzed on day 3. Crosslinked, sheared chromatin was precipitated by goat anti–IRF-8, rabbit anti-PU.1 antibody, or control IgG (normal goat IgG for IRF-8 ChIP and normal rabbit IgG for PU.1 ChIP). Precipitated DNA was analyzed in duplicate by real-time PCR to detect genomic DNA from the cystatin C and cathepsin C promoter regions using primers that amplified the IECS region of each promoter. Levels of precipitated promoter DNA (ChIP signals) are shown as values relative to 0.1% input DNA (mean ± standard deviation). TheHPRT promoter was analyzed as a control irrelevant to IRF-8 and PU.1. (B) Binding of endogenous IRF-8 and PU.1 to the target gene promoters detected by ChIP assay. Peritoneal macrophages from wild-type mice were analyzed as in panel A. (C) ChIP assay for binding of IRF-8 and PU.1 to the IECS in vivo. Tot2 cells were transduced with SIRV-IECS-Ld40-GFP, and then with MSCV or MSCV–IRF-8. Cells were analyzed on day 3 after transduction of MSCVs. The IECS DNA was detected using primers designed to amplify the IECS sequence in SIRV.