After the appropriate treatment, the rats were killed with CO2. The retinas were dissected and homogenized by sonication at 4°C. Each retina was homogenized in 200 μL of lysis buffer (5 mM HEPES [pH 7.5], 50 mM NaCl, 0.5% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM benzamidine, phosphatase inhibitor cocktails 1 and 2 (10 μL/mL; Sigma, St. Louis, MO), and proteinase inhibitor cocktail set III (10 μL/mL; Calbiochem, San Diego, CA). The insoluble pellet was removed by centrifugation at 4°C, and the protein concentration of the supernatant was measured with a protein assay reagent kit (Bio-Rad Laboratories). Soluble protein (80 μg) was resolved by SDS-PAGE on 4% to 12% 1.0-mm 10-well Bis-Tris minigels (NuPAGE Novex; Invitrogen, Carlsbad, CA) and electrotransferred to a 0.2-μm pore PVDF membrane (Invitrogen). The membrane was then cut into two pieces, and the upper and lower portions of the membrane were blotted with monoclonal anti-β-actin 1:10,000 (Catalog SC-47778 HRP; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF 1:200 (Catalog SC-7269; Santa Cruz Biotechnology) antibodies, respectively. Immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer, Waltham, MA), and relative band density was determined on scanned autoradiographs with image-analysis software (FluorChem HD2; Alpha Innotech Corp., San Leandro, CA). The intensity of the β-actin signal, which was not different in diabetic and control rats (not shown), was used as an endogenous control for loading. Data are expressed as the VEGF:β-actin densitometric unit ratio.