(a) Tissue survey of Foxo isoform expression in mice. We hybridized multiple tissue Northern blots (filter membranes; Clontech Laboratories Inc.) with probes encoding the three Foxo isoforms, labeled with ³²P at similar specific activities to obtain readily comparable hybridization signals. (b) Foxo1 expression in cultured β cells. We isolated mRNA from SV40-transformed mouse hepatocytes (lane 1), βTC-3 (lane 2), and HEK293 cells (lane 3), and performed Northern blot analysis using specific probes for Foxo1, Foxo3, and Foxo4. (c) Time course analysis of Foxo1 expression in islets. We isolated mRNA from islets of wild-type mice at 2, 4, 8, and 24 weeks of age. We performed RT-PCR using primers for Foxo1, Foxo3, Foxo4 (not shown, because no amplification could be seen), Irs2, Pdx1, insulin, glucagon, pancreatic polypeptide, and β-actin as indicated in Methods. (d) Islet immunohistochemistry. We performed double immunohistochemistry with anti-Foxo1 and anti-insulin antisera on the same section to localize Foxo1 expression. We visualized the anti-insulin antibody with CY3-conjugated anti–guinea pig IgG, and the anti-Foxo1 with FITC-conjugated anti-rabbit IgG.