Fig. 1.

CRTC2 increases CREB promoter occupancy in response to fasting signals. (A) Immunoblot (Upper) and immunocytochemical (Lower) analysis showing CRTC2 dephosphorylation and nuclear translocation in primary hepatocytes exposed to forskolin (FSK). Scale bar, 5 um. (B) Chromatin immunoprecipitation (ChIP) assay of CREB (Upper) and CRTC2 (Lower) occupancy over the human PEPCK promoter in HEK293T cells exposed to FSK (black bars) or vehicle (white bars) (n = 3, P < 0.05; SEM). Location of CREB binding sites indicated. (C) Gel mobility shift assay of CREB and GST-CRTC2 proteins using a ³²P-labeled double-stranded CRE oligonucleotide. Protein–DNA complexes and free probe indicated. (D) (Upper Left) Schematic diagram showing organiziation of wild-type or mutant CRTC2 vectors lacking either the N-terminal CREB binding domain (aa 1–50) or central regulatory region (aa 51–539) indicated. (Upper Right) Immunoblot with anti-flag epitope antiserum showing relative expression of each CRTC2 polypeptide in transfected cells. (Lower) Transient assay of HEK293T cells transfected with cAMP responsive EVX-luc reporter vector and exposed to FSK as indicated (n = 3, P < 0.05; SEM).