Figure 6.

Identification of the Stat binding site in the TRH promoter. (a) 293T cells were cotransfected with the indicated hTRH constructs. Leptin responsiveness was then determined. The data are expressed as percent of maximal response where the fold stimulation of the –150 construct is set at 100%. (b) EMSA was performed with a radiolabeled probe spanning the human Stat site and nuclear extract from leptin-treated or untreated cells. The specificity of the bound complex was discerned by using an anti-Flag (αF) antibody. (c) A similar EMSA paradigm was used on the canonical SIEm67 probe. (d) A similar EMSA paradigm was used on a probe spanning the murine Stat element. (d) EMSA was performed on the murine Stat element with the addition of cold oligonucleotides representing the murine (lanes 3 and 4) or human (lanes 5 and 6) Stat elements. A 10× cold competitor was added in lanes 3 and 5, and 100× in lanes 4 and 6.