Figure 1. Recruitment of SREBP-1c to the FASN promoter and lack of binding to the GCK promoter in hepatocytes induced for SREBP-1c expression by T0901317.

Primary rat hepatocytes were placed in culture as described in the Experimental procedures section. After 8 h, the hepatocytes were stimulated with 10 μM T0901317 and culture was continued in the presence of T0901317 for 17 h. Where indicated, cells were induced with 30 nM insulin in addition to T0901317 for the last hour of treatment. Other hepatocytes were treated with insulin alone for 1 h. The cells were then harvested for ChIP assay of SREBP-1c binding to the FASN and GCK promoters (panels A and B) or for immunoblot analysis of SREBP-1c precursor and processed forms (C). (A) FASN and GCK promoter products and GCK intron product after PCR amplification of ChIP with anti-SREBP1 antibody. The specific gene regions were amplified by PCR and products were electrophoresed in agarose gels containing ethidium bromide. The images represent gels visualized by UV transillumination. (B) Enrichment of the FASN promoter (left-hand panel) but not the GCK promoter (right-hand panel) in chromatin precipitated with anti-SREBP1 antibody after the indicated cell treatments. Enrichment factor was calculated as described in the Experimental procedures section. Results are means±S.E.M. of three separate experiments. Control immunoprecipitates generated with normal rabbit IgG did not reveal significant enrichment of either promoter under any condition (results not shown). (C) Immunoblot of SREBP-1c precursor (upper panel) and processed forms (lower panel) in hepatocytes under various treatments. The experiment was performed three times with similar results. kd=kDa.