Fig. 3.

GH-induced recruitment of p300 to c-Fos depends on an intact phosphorylation site at T188 of C/EBPβ. A, Plasmids for shβ, and for rescue constructs expressing WT or T188A C/EBPβ, as well as for GHR, were expressed in 293T cells; 48 h later, cells were treated with GH for 30 min. Nuclear extracts were analyzed by ChIP using antibodies against p300 or C/EBPβ and probed for the c-Fos C/EBP site. Control without antibody and 1% input are shown. B, Plasmids for shβ and rescue constructs for WT or T188A C/EBPβ, or vector, were transiently transfected into 293T cells, with (black bar) or without (open bar) plasmid for CMV-p300. Plasmids for Fos-luc and RSV-β-gal were also transfected; 48 h later, cells were lysed and used for luciferase assay. The c-Fos promoter activity in cells expressing control pcDNA3 without p300 is set equal to 1. Each bar shows mean ± se for three independent experiments. In C/EBPβ-deficient cells transiently expressing shβ, significant coactivation of c-Fos-luc by p300 occurs with rescue of WT but not T188A C/EBPβ.