Pten a2b1

Protein extraction was performed using a lysis buffer containing a protease inhibitor cocktail (Complete; Sigma-Aldrich), and protein quantification was performed using the bicinchoninic acid method (Pierce; Rockford, IL). 50 μg of protein per condition was run on SDS-PAGE gels and then transferred onto PVDF membranes (BioRad), which were subsequently blocked for 1 h with a solution of 5% skim milk/in TBS/Tween 0.1%. Membranes were incubated overnight with primary antibodies against PTEN (A2B1) (diluted 1:250; Cat No. 7974, Santa Cruz Biotechnology), AKT (diluted 1:5000; Cat No. 4691, Cell Signaling Technology), phospho-AKT (diluted 1:1000; Cat No. 9271, Cell Signaling Technology), β-Actin (diluted 1:10,000; Cat No. A3854, Sigma Aldrich), ERK1/2 (diluted 1:5000; Cat No. 9102S, Cell Signaling Technology) and pERK1/2 (diluted 1:500; Cat No. 9106S, Cell Signaling Technology). Subsequent washes were performed with TBS/Tween 0.1% and then membranes were incubated for 1 h with anti-rabbit and anti-mouse secondary antibodies diluted 1:3000 (Invitrogen), followed by washes with TBS/0.1% Tween. The presence of the proteins was revealed by chemiluminescence (Perkin-Elmer). Images were captured and analyzed using a UVP BioImaging System and LabWorks Image Acquisition and Analysis Software (UVP, Inc. Upland, CA).