2.7. Western blot analysis and antibodies

Total protein lysates (20 μg) were resolved on a 4–12% bis-tris gel and transferred to Immobilon PVDF transfer membranes. Membranes were incubated for 40 min at room temperature in blocking solution (TRIS-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20), followed by an overnight incubation in primary antibodies at 4 °C. Membranes were then washed 3 times and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h. After 3 additional washes, the immune complexes on the membranes were visualized by ECL detection. The following antibodies were purchased and utilized in our study: Cell Signaling (Danvers, MA): phospho-Akt (Ser473) (Western blot; #4058; 1:1000), total Akt (Western blot; #2920; 1:1000), PARP (Western blot; mAb #9532; 1:1000). BD (San Jose, California): phospho-Akt (Immunohistochemistry; Ser473; 1:100). Abcam (Cambridge, MA): Akt2 (Immunohistochemistry; #55391; 1:500), cyclin D1 antibody [EPR2241] (ab134175). Mouse monoclonal anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO).