Chemiluminescence imager

Retinal homogenates were prepared in ice–cold lysis buffer supplemented with PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (all from KeyGen BioTECH, Nanjing, China) with a tissue homogenizer and cleared via centrifugation (12,000× g for 10 min at 4 °C). The retinal protein concentration was determined using bicinchoninic acid (BCA) kit (KeyGen BioTECH), and equal amounts of total protein were electrophoresed on 10% polyacrylamide gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) following a standard protocol. After blocking for 10 min with QuickBlock™ Blocking Buffer (Genscript, Nanjing, China), the membranes were incubated with primary antibodies for 4 °C overnight followed by incubation with HRP–conjugated secondary antibodies for 2 h at room temperature. After washing three times with 0.1% Tween–20/TBS, the immunoreactive proteins were visualized with an enhanced chemiluminescence kit (Millipore) and a chemiluminescence imager (Tanon Science & Technology, Shanghai, China). For stripping, the membranes were incubated in a mild stripping buffer (Epizyme Biomedical Technology, Shanghai, China) for 10 min when necessary. The recorded data and densitometric analysis were quantified using ImageJ. The relative expression of a specific protein was calculated with respect to β–Actin.

Orbital tissue and OFs were lysed to extract protein with a protein extraction kit (KeyGEN, Nanjing, China). The concentration was measured using a BCA (Cwbiotech, Beijing, China). Protein lysates were electrophoresed, transferred to membranes, blocked and incubated with primary and secondary antibodies. Finally, images were obtained with a chemiluminescence imager (Tanon Science & Technology, Shanghai, China). All antibodies were purchased from Cell Signaling Technology.