Western Blot Analysis of PI3K/Akt Signaling

Whole-cell lysates from RPMI-8226 cells were prepared. The amount of cellular protein was measured by the method proposed by Lowry et al31 (link) and stored at −80°C prior to use. Twenty microgram of protein of each sample was separated on SDS-PAGE gels as previously described.32 (link) After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with Tris-buffered saline 0.05% Tween-20 containing 5% skim milk or 3% bovine serum albumin (Sigma, St Louis, MO, USA), blots were incubated at 4°C overnight with the primary rabbit polyclonal or monoclonal antibodies against human PI3K (1:1,000), Akt (1:500), phospho-Akt (pSer⁴⁷³, 1:500), Caspase-3 (1:2,000), Bcl-2 (1:500), and Bax (1:500) (Cell Signaling Technology, Danvers, MA, USA), respectively. Membranes were reprobed with anti β-actin antibody (1:1,000; Sigma, St Louis, MO, USA) as loading control. Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit or mouse immunoglobulins were used as a secondary antibody (Sigma, St Louis, MO, USA). The bounded antibody was detected by enhanced chemiluminescence using Immobilon Western reagents (Millipore, Billerica, MA, USA) and exposed to X-ray film according to the manufacturer’s protocol.