T erk1 2

For the western blot analysis, the neural retinas were rapidly dissected from the euthanatized rats and lysed in RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor (Beyotime, Shanghai, China) and phosphatase inhibitor (Roche, Mannheim, Germany). Aliquots containing 30 μg of protein were separated by SDS-polyacrylamide gel electrophoresis using a 10% gel, and the separated proteins were blotted onto PVDF membranes (Millipore, Billerica, MA) in a wet transfer unit (Bio-Rad, Hercules, CA). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: GPR91 (1:1000), p-ERK1/2 (1:3000) , t-ERK1/2 (1:3000) , COX-2 (1:200), anti-VEGF (1:200, Abcam, Cambridge, MA) andβ-actin (1:1000, Abcam). After being washed with TBS-Tween 20, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (1:1000, ProteinTech Group, Chicago, IL) for 1 h at room temperature. The bands were visualized using an enhanced ECL detection kit (Pierce Biothechnology, Rockford, IL). For the ELISA, the vitreous fluid samples were collected from rat eye for enzyme-linked immunosorbent assay analysis using kits from R&D Systems (Minneapolis, MN) following the instructions provided by the manufacturer.

Protein was collected and quantified at 48 h after transfection of the CRISPR plasmid. Same amount of total proteins were loaded to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The primary antibodies against HPV16-E7 (1:1000; Biorbyt, Shanghai, China), retinoblastoma (RB; 1:800; Proteintech, Rosemont, IL), cyclin-dependent kinase 2 (CDK2; 1:500; Proteintech), E2F transcription factor 1 (E2F1; 1:1000; Proteintech), total-Akt (t-Akt; 1:800; Abcam, Shanghai, China), phosphorylated Akt (p-Akt; 1:800; Abcam), total extracellular signal-regulated kinase 1/2 (t-ERK1/2; 1:500; Abcam), or phosphorylated-ERK1/2 (p-ERK1/2; 1:500; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000; Proteintech) were used. Then, blots were incubated with horseradish-peroxidase-conjugated secondary antibodies, and the bands were visualized using chemiluminescence. The experiments were repeated at least 3 times.