Quantification of Retinal Endothelial Proteins

Total proteins from the treated retinal endothelial cells were extracted with RIPA lysis buffer (Beyotime; cat.: P0013C) containing PMSF (1:100; Beyotime; cat.: ST506). The protein concentration was quantified by BCA assay (Sangon biotech; cat.: C503061) according to the manufacturer’s instruction, and further adjusted to 2 mg/ml before dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). When the SDS-PAGE gel electrophoresis was completed, the proteins were transferred onto a nitrocellulose filter membrane (NC; Millipore; cat.: HATF00010). After blocking with 5% milk in PBST buffer for 1 h, the membrane were incubated with the primary antibodies anti-STAT3 (1:1000; Cell Signaling Technology; cat.: 9139S), anti-p-STAT3 (1:1000; Cell Signaling Technology; cat.: 9145 T), anti-HIF-1α antibody (1:1000; Proteintech; cat.: 20,960-1-AP), anti-VEGFA (1:1000; Abcam; cat.: ab1316) and anti-α-tubulin (1:8000; Abcam; cat.: ab18207) at 4°C overnight. After that, the membrane was incubated with the corresponding secondary antibody conjugated with HRP (1:1000; Beyotime; cat.: A0208) for 2 h at room temperature. After rinsing with PBST buffer for three times, the immunoreactivity of membrane was visualized using the SuperSignal West Pico kit (Pierce) [20 (link)].