FIGURE 3.

FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in immortalized gonadotrope cells. A, Western blot analysis was performed on whole cell extracts from the indicated cell lines using a FOXO1 primary antibody and a horseradish peroxidase-linked secondary antibody. β-Tubulin was used as a loading control. B, LβT2 cells were incubated overnight in serum-free media, pretreated for 1 h with vehicle (Veh) or 50 μm LY294002 (LY), and then treated for 10 min with vehicle, 1–100 nm insulin, 50 μm LY, or 10 nm insulin and 50 μm LY, as indicated. Western blot analysis was performed using FOXO1 serine 256 (P-FOXO1), total FOXO1, and β-Tubulin antibodies. C, LβT2 cells were incubated overnight in serum-free media, pretreated for 30 min with vehicle (Veh), or 50 μm LY294002 (LY), and then treated for 30 min with vehicle, 10 nm insulin, 50 μm LY294002, or 10 nm insulin and 50 μm LY294002, as indicated. IF was performed with a FOXO1 primary antibody and an Alexa594-conjugated goat anti-rabbit secondary antibody. Representative images were obtained using a Nikon Eclipse TE 2000-U inverted fluorescence microscope at 60× magnification.