Foxo1 binds to the Pdx1 promoter and inhibits Foxa2-induced Pdx1 transcription. (a and b) Gel shift assays. (a) We used nuclear extracts from kidney epithelial LLC cells transduced with Foxo1 adenovirus to test the specificity of binding to the Pdx1 probe. We chose this cell type because it does not express Foxa2 (13), which is abundant in βTC-3 cells. The concentrations of cold competitor used are indicated by the triangle and are 0, 100-, 300-, and 900-fold excess for both wild-type and mutant probes. (b) Antibody-induced supershift. We incubated βTC-3 nuclear extracts with antisera against Foxo1 and Foxa2 or with nonimmune serum (control). I and II correspond to the two main complexes detected in the absence of antiserum, III and IV to the supershifted complexes. (c) Foxa2, but not Foxo1, induces Pdx1 promoter activity. We cotransfected βTC-3 cells with the plasmids indicated at the bottom of the graphs. After 48 hours, we measured luciferase activity and normalized it by SEAP activity relative to the basic pCMV5 vector. (d) Foxo1 inhibits Foxa2-dependent Pdx1 transcription. We cotransfected βTC-3 cells with the Pdx1-luciferase reporter gene and pCMV5 vector or pCMV5-Foxa2. Five hours after transfection, cells were transduced with control adenovirus or with adenovirus encoding ADA-Foxo1 at the indicated moi. After 36 hours, we measured relative luciferase activity. (e) We determined immunoreactive Foxo1 and Foxa2 levels by Western blotting in cells transduced with Foxo1 at different moi’s. Foxo1 was not detectable by this approach in untransduced cells because of its low expression levels.