Foxo3

Tissues and cells were kept in ice‐cold RIPA buffer with protease (Sigma, St. Louis, MO, USA) inhibitor to extract total proteins at 24 hr after treatment. Proteins were separated on a 12% SDS‐PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were then blocked in 5% nonfat milk with Tris‐buffered saline and 0.1% Tween 20 at room temperature for 2 hr. Primary antibodies were incubated at 4°C for at least 12 hr, including LC3B (1:1,000, Cell Signaling Technology, Beverly, MA, USA), SQSTM1 (1:1,000, Cell Signaling Technology, Beverly, MA, USA), FOXO3 (1:1,000, Cell Signaling Technology, Beverly, MA, USA), ATG7 (1:1,000, Cell Signaling Technology, Beverly, MA, USA), and GAPDH (1:1,000, Santa Cruz Biotechnology, CA, USA). Membranes were subsequently incubated with HRP‐conjugated secondary antibodies for another 2 hr at room temperature. Immunoblots were visualized by an enhanced chemiluminescence detection kit (ECL kit; Millipore, Billerica, MA) under a chemiluminescence imaging analysis system (Amersham Imager 600, GE, CT, USA). Relative integrated density values were calculated using Image J software.

Cells were lysed by 1x laemmeli buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 2.5% β-mercaptoethano, and 0.01% bromophenol blue, followed by boiling at 95°C for 10 min. Total cell lysates were separated in SDS polyacrylamide gels and then electrophoretically transferred to PVDF membranes that were pretreated with 100% methanol for 5 minutes. The prepared membranes were blocked with 3% skim milk for 1 hour at room temperature and then sequentially incubated with indicated primary antibody overnight at 4°C and secondary antibody for 1 hour at room temperature. The used primary antibodies included rabbit monoclonal antibodies to FoxO1 (Cell Signaling Technology, 2880), FoxO3 (Cell Signaling Technology, 2497), cleaved caspase 3 (Cell Signaling Technology, 9664), β-actin (ABclonal, AC026), and a mouse monoclonal antibody to ORFK8 (Santa Cruz, F33P1). β-actin was used as the loading control.