Retinas (n = 45) were sonicated in 10 mM Tris-HCl (pH 7.6) containing 5 mM EDTA, 3 mM EGTA, 250 mM sucrose, protease, and phosphatase inhibitor cocktails and centrifuged at 22,000g for 30 minutes at 4°C. The supernatants were used to detect VEGF, HIF-1α, STAT3, pSTAT3, albumin, Bax, Bcl-2, and cytochrome c. Pellets were resuspended in 20 mM HEPES, pH 7.4, containing 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 4 mg·mL−1 n-dodecyl-β-maltoside, protease, and phosphatase inhibitor cocktails, and centrifuged at 22,000g for 30 minutes at 4°C. The supernatants were used to detect VEGFR-2, pVEGFR-2, and occludin. Protein concentration was determined using a fluorometer (Qubit; Invitrogen, Carlsbad, CA, USA). Aliquots of each sample containing equal amounts of protein (30 μg) were subjected to SDS-PAGE. We used β-actin as the loading control. The gels were transblotted onto PVDF membrane, and the blots were blocked in 3% skim milk for 1 hour at room temperature. Blots were then incubated overnight at 4°C with rabbit polyclonal antibodies directed to VEGF (1:200); VEGFR-2 (1:200); STAT3 (1:200); albumin (1:200); occludin (1:250); Bax (1:100); and Bcl-2 (1:100); or mouse monoclonal antibodies directed to pVEGFR-2 (1:100); HIF-1α (1:200); pSTAT3 (1:200); and cytochrome c (1:500). The same membrane was reblotted with a mouse monoclonal antibody directed to β-actin (1:10,000). Finally, blots were incubated for 1 hour at room temperature with a mouse anti-rabbit horseradish peroxidase–labeled secondary antibody (1:5000) or a rabbit anti-mouse horseradish peroxidase–labeled secondary antibody (1:25,000) and developed with the enhanced chemiluminescence reagent. Images were acquired (ChemiDoc XRS+; Bio-Rad Laboratories, Inc.), and the optical density (OD) of the bands was evaluated (Image Lab 3.0 software; Bio-Rad Laboratories, Inc.). The data were normalized to the level of β-actin, VEGFR-2, or STAT3, as appropriate. All experiments were run in duplicate. After statistical analysis, data from the different experiments were plotted and averaged on the same graph.