Western blot analysis of AKT [pS473]. Experiment was performed with 20 µg of cell lysates from NIH/3T3 (lane 1), NIH/3T3 treated for 10 min with 25 ng/mL of PDGF (lane 2), and U-87 MG (lane 3), and using an Invitrogen™ NuPAGE™ 4-12 % Bis-Tris gel (Cat. No. NP0321BOX), Invitrogen™ XCell SureLock™ Electrophoresis System (Cat. No. EI0002), Invitrogen™ Sharp Pre-Stained Protein Standard (Cat. No. LC5800), and Invitrogen™ iBlot™ Dry Blotting System (Cat. No. IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hr at room temperature. AKT [pS473] was detected (~55 kDa) using ABfinity™ AKT [pS473] recombinant rabbit monoclonal antibody (Cat. No. 700392) at 1–3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the blot on the right. Goat anti-rabbit IgG-HRP secondary antibody (Cat. No. G21234) at 1:5,000 dilution was used, and chemiluminescent detection was performed using the Invitrogen™ ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005).