Ser249-FOXO1 is less affected by AKT inhibition. A–F, GFP-FOXO1, GFP-FOXO1–S249A, or GFP-FOXO1–S249D was transfected in primary cortical neurons treated with DMSO or LY294002 for 16 h. Representative images and the quantification of subcellular localization of GFP-FOXO1, GFP-FOXO1–S249A, and GFP-FOXO1–S249D were shown in top and bottom panels, respectively. G–I, GFP-FOXO1-AAA, GFP-FOXO1-AAA-S249A, or GFP-FOXO1-AAA-S249D was transfected in primary cortical neurons. Representative images and the quantification of subcellular localization of GFP-FOXO1-AAA, GFP-FOXO1-AAA-S249A, and GFP-FOXO1-AAA-S249D were shown in top and bottom panels, respectively. J–K, Primary cortical neurons were transfected with GFP-FOXO1-AAA, GFP-FOXO1-AAA-S249A, or GFP-FOXO1-AAA-S249D plasmids together with the 3xIRS-luciferase (J) or Bim-luciferase (K) reporter gene and subjected to luciferase assays. Data are mean ± SEM; n = 5. *p < 0.05.