(v/v). The homogenates were centrifuged at 5,000 g for 30 min at 4uC. The supernatant was collected,and protein concentration was measured according to the Bradford [54] method, using bovine serum albumin as standard. The protein samples were stored at 280uC.Protein samples were dissolved in 46sample buffer (pH 6.8) containing: Tris–HCl, 250.0 mM; sucrose, 200.0 mM; dithiothreitol, 300.0 mM; Coomassie brilliant blue-G, 0.01%; and sodium dodecyl sulfate, 8% and denatured at 95uC for 5 min, then the equivalent amounts of proteins (30 mg) were separated by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 mm Polyviny lidene fluoride(PVDF) membrane.In addition, the gels stained with Coomassie blue were used to confirm the equal amounts of protein loaded on each lane. The membranes were incubated overnight at 4uC with the following primary antibodies: primary polyclonal rabbit anti- PI3K-p110c antibody(1:100), primary polyclonal rabbit anti-p- AKT or anti-AKT antibody (1:1000), primary polyclonal rabbit anti-p-ERK1/2 or anti-ERK1/2 antibody (1:1000), or primary polyclonal rabbit-anti-GAPDH antibody (1:10,000). The specific- ity for these antibodies was confirmed by loss of bands in the absence of primary antibodies.The membranes were extensively washed with Tris-buffered saline Tween20 and incubated for 1 hour with the secondary antibody conjugated with Horseradish peroxidase (HRP) (1:10,000) at room temperature. The immune complexes were detected by using ECL western detection reagents. Western blot densitometry analysis of signal intensity was performed using Quantity One 4.6.2 (Bio-Rad; Hercules, CA) and phosphorylation levels of AKT from densitometry were normalized to AKT. The blot density from control groups was set as 100%. References 1. 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