Total protein was extracted from cells using lysis buffer (Roche Diagnostics, Basel, Switzerland). Protein samples (30 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) ultrafiltration membrane (Sigma-Aldrich) for 2 h at 4°C. The membranes were blocked with 5% nonfat milk for 1 h at room temperature. The membranes were washed three times for 5 min each with 15 ml TBS Tween 20 (TBST; Cell Signaling Technology). The membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated antibody for 2 h at 37°C. Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL) blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and GAPDH served as the internal reference. The primary antibodies used in this study are as follows: GSN, PI3K, Akt, p-Akt and GAPDH antibody (1:1000; Cell Signaling Technology). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Santa Cruz Biotechnology).