Western Blot Analysis of FoxO Proteins

Western blotting was performed as described elsewhere [15 (link)]. Briefly, 10 μg cytoplasmic or nuclear protein was separated on SDS-PAGE and transferred to PVDF membranes. Membranes with nuclear protein were incubated with anti-FoxO3a (#12829) or anti-FoxO1 (#2880). Membranes with cytoplasmic protein were incubated with anti-p-FoxO3a (#2599), anti-p-FoxO1 (Ser256) (#9461), anti-p-AKT (Ser473) (#9271), anti-p-AKT (Thr308) (#4056) or anti-AKT (#9272; Cell Signaling Technology, Danvers, MA, USA) antibodies at 4°C overnight diluted 1:1000 in Solution 1 (TOYOBO, Co., Ltd., Osaka, Japan). After TBST washes, membranes were incubated with HRP-conjugated sheep anti-rabbit IgG (GE Healthcare Life Science, Piscataway, NJ, USA) diluted 1:2000 in Solution 2 at room temperature for 60 min. Internal controls were mouse anti-Hsc70 (sc-7298; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for cytoplasmic immunoblotting and mouse anti-Histone H3 (#4499) (Cell Signaling Technology, Danvers, MA, USA) for nuclear immunoblotting. Hsc70 and Histone H3 were used for normalization.