Trib2 is a direct target gene of Notch1. (A) Reverse transcriptase (RT)–PCR analysis of Trib2 expression in Scid.adh cells treated with GSI (+) or DMSO as vehicle control (−). Cells were transduced with an empty vector expressing only the NGFR selection marker (NGFR) or a vector expressing ICN1 (ICN1). Trib2 mRNA expression was assessed. Hprt is an internal loading control. Results are representative of triplicate experiments. (B) Schematic representation of the Trib2 promoter region. Four putative CSL-binding sites were identified in the region shown, including a tandem CSL site at − 6 kb and 3 single binding sites at − 22 kb, − 6.1 kb, and − 3 kb relative to the translational start site. (C) Notch1 binds to CSL-binding sites in the Trib2 promoter. Chromatin immunoprecipitates were performed on cross-linked fragmented DNAs prepared from Scid.adh cells. Immunoprecipitations were carried out with antibodies against Myc as a negative control (control), acetylated histone 4 (AcHIS4), and Notch1. PCR was performed using primers directed against indicated CSL-binding site regions at − 22, − 6, and − 3 kb from the ATG translational start site of Trib2. PCR for the Hes1 promoter region is shown as a positive control. The figure shown is representative of duplicate experiments. For the − 6-kb region, which shows the strongest enrichment, results are representative of duplicate samples and triplicate experiments. (D) After ChIP as described in panel C, using either Notch1 or normal rabbit control IgG antibody, RQ-PCR was performed using primer sets flanking putative CSL-binding sites in the Trib2 promoter. RQ-PCR was performed using the primers at − 6 kb to quantify enrichment in this region, and also using primers flanking a conserved CSL-binding site at − 6.1 kb. Graphs represent mean of the ratios of the amount of IP DNA/input from values of duplicate wells plus or minus standard deviation. Data are representative of 3 independent experiments.