Retinas were homogenized in lysis buffer (50 mM Tris [pH 7.8], 100 mM NaCl, 5 mM EDTA, 0.05% SDS, 1% TX-100, 2.5% glycerol, and 1 mM PMSF). Protein concentration was determined using the Bradford assay kit (Bio-Rad). Equal amounts of total protein were separated using 10% SDS-PAGE, and electrotransferred to PVDF membrane. Membranes were blocked with 5% nonfat milk in TBST, and incubated in anti-VEGF-A (sc-57496; Santa Cruz Biotechnology, Santa Cruz, CA), anti-VEGFR2 (sc-6251; Santa Cruz Biotechnology), or antipigment epithelium-derived growth factor (PEDF, ab14993; Abcam, Cambridge, MA) for 1 hour at room temperature. After washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies, and visualized in accordance with the manufacturer's guidelines (SuperSignal West Dura Extended Duration Substrate; Thermo Fisher Scientific, Waltham, MA). Imaging and densitometric analysis were conducted using a commercial imaging system (Kodak Image Station 4000R; Carestream Health, Inc., Rochester, NY) and the pixel densities in each band were normalized to the amount of β-actin in each lane.