Anti phospho akt ser473 d9e

Cell extracts were made by lysing PBS washed cell pellets in radio-immunoprecipitation assay buffer (RIPA) supplemented with protease inhibitors (Complete protease inhibitor, Roche Diagnostics). Following incubation on ice, clear lysates were obtained by centrifugation. Protein concentrations were determined by Bradford's assay (Bio-Rad). For each sample, 30 μg of protein was loaded on each gel. Proteins were transferred onto a PVDF membrane using a tank blotter (Bio-Rad). The membranes were then blocked with 5% milk and 1X Tris buffered saline plus Tween 20 (TBST) and incubated with primary antibody overnight at 4°C. Membranes were then washed with 1X TBST and incubated with the corresponding secondary antibody. Membranes were again washed with 1X TBST, incubated with chemiluminescent substrate according to manufacturer's protocol (SuperSignal, Pierce) and visualized by autoradiography. The antibodies used include anti-PTEN (559600, BD Pharmingen), anti-phospho PTEN (9554, Cell Signaling), anti-TBX2 (C-17, Santa Cruz Biotechnology), anti-TBX2 (gift of C. Goding, University of Oxford), anti-TBX3 (A-20, Santa Cruz Biotechnology), anti-AKT (pan, C67E7, Cell Signaling), anti-phospho-AKT (Ser 473 D9E, Cell Signaling) and anti-GAPDH (6C5, Millipore). At least three biological replicates were performed for each experiment.