Fig. 6.

C/EBPβ occupies predicted C/EBP sites on the Cyr61 promoter. A, 3T3-F442A preadipocytes stably expressing shβ or control sh-C cells were treated without (lanes 1 and 3) or with GH (lanes 2 and 4) for 30 min. Nuclear extracts were analyzed by ChIP using antibodies against C/EBPβ or acetylated H4 (AcH4), or IgG, with probes for the C/EBP site of c-Fos or the two predicted sites on Cyr61; 1% input is also shown. B, Liver from C/EBPβ deficient (KO) or control (WT) mice was used to prepare nuclear extracts for ChIP. Samples were analyzed as in A. C, Diagram of Cyr61 promoter shows locations (bars) of predicted C/EBP-CREB motifs that are conserved in mouse and human sequences (see Table 2). Diagram is not to scale. To test Cyr61 promoter activation, plasmid for Cyr61-luc (0.5 μg/well) containing the predicted C/EBP sites, and the indicated amounts of plasmid for C/EBPβ (ng/well) and RSV-β-gal (20 ng/well), were transfected into 293T cells in 24-well plates; 48 h later, lysates were analyzed for luciferase activity. Each bar shows the mean of three to five independent experiments. RLU, Relative luciferase units.