After cervical dislocation, the eyes were enucleated, and the retinas were removed and rapidly frozen in liquid nitrogen. To extract the proteins, the tissue was homogenized in cell lysis buffer with a homogenizer (Microtec Co., Ltd., Chiba, Japan). The lysate was centrifuged at 12,000g for 20 minutes, and the supernatant was used for the Western blotting. The protein concentration was measured by comparison with known concentrations of BSA with a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). Equal parts of an aliquot of protein and sample buffer with 10% 2-mercaptoethanol were used to separate the proteins by 15% SDS-PAGE. The separated protein was then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Merck Millipore). The membranes were blocked for 30 minutes at room temperature with 5% Block One-P (Nacalai Tesque, Inc., Kyoto, Japan) in 10 mM Tris-buffered saline with 0.05% Tween 20, and then incubated overnight at 4°C with the primary antibody. The primary antibodies used were rabbit anti-PDGFR-β (1:200; Santa Cruz Biotechnology), rabbit anti-phospho PDGFR-β (1:200; Abcam, Cambridge, MA, USA), rabbit anti-PDGF-BB (1:400; Abcam), rabbit anti-Akt (1:1000; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-phospho Akt (Ser473, 1:1000; Cell Signaling Technology, Inc.), and mouse anti-β-actin (1:5000; Sigma-Aldrich Corp., St. Louis, MO, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Pierce Biotechnology, Rockford, IL, USA) and HRP-conjugated goat anti-mouse IgG (1:2000; Pierce Biotechnology) used for 1 hour at room temperature. The immunoreactive bands were made visible with Immunostar LD (Wako Pure Chemical, Osaka, Japan) and then measured with the LAS-400 Mini digital imaging system (Fuji Film Co., Ltd., Tokyo, Japan).