Robo4

For in vivo experiments, the retinal tissues from control or diabetic rats were isolated and washed in PBS twice. For in vitro experiments, total protein was collected from HREC or ARPE-19 cells cultured under hyperglycemia. The tissues or cells were lysed for 60 min on ice in Total Protein Extraction Buffer with protease inhibitor (Transgen Biotech, Beijing, China) according to the manufacturer’s protocol and then sonicated. The lysates were collected at 12,000 rpm for 10 min at 4 °C. Protein concentrations were evaluated using a Bicinchoninic Acid Protein Assay Kit (Beyotime, Jiangsu, China). Protein lysates were electrophoresed on 10% SDS polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore, USA), and then blocked in 5% skim milk for 1 h. Primary antibodies against VEGF (1:500, Affinity, USA), Robo4 (1:50, Abcam, USA) and β-actin (1:1000, CMC-TAG, USA) were applied separately at 4 °C overnight. Blots were then treated with secondary antibodies (1:5000; Boster, China) for 40 min. Finally, an Enhanced Chemiluminescence (ECL) Plus Kit (Millipore, USA) was applied for visualization. The gray bands were calculated using the Image J software.