Cw0100

Total proteins were extracted with RIPA lysis buffer (Servicebio Technology, Wuhan, China). Thereafter, proteins were separated via SDS–polyacrylamide gel electrophoresis and then were subsequently transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were incubated with primary antibodies for the following proteins: Akt (1 : 1000; 9272S, CST), p-Akt (Ser473; 1 : 1000; 4060S, CST), Rps6kb1 (1 : 1000; 9202S, CST), p-Rps6kb1 (Thr-389; 1 : 1000; 9205S, CST), Eif4ebp1 (1 : 1000; 9452S, CST), p-Eif4ebp1 (Ser-65; 1 : 1000; 9451S, CST), Yap (1 : 1000; 14074S, CST), p-Yap (Ser-127; 1 : 1000; 13008S, CST), Gapdh (1 : 2000; CW0100, CWBiotech, Beijing, China), and β-Actin (1 : 2000; CW0096, CWBiotech, Beijing, China). The membranes were then incubated with the corresponding secondary antibody (goat anti-rabbit IgG: 1 : 4000; BF03008, Biodragon-immunotech, Beijing, China; goat anti-mouse IgG: 1 : 4000, BF03001, Biodragon-immunotech, Beijing, China) for 2 h. Finally, the immunoblots were visualized with an ECL kit (CWBiotech, Beijing, China). The optical density of phosphorylated proteins (p-AKT, p-Rps6kb1, p-Eif4ebp1, and p-Yap) was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized by the amount of the loading control on the same membrane.

To perform Western blot analysis of whole retinae, the retinal samples were first lysed in ice-cold RIPA lysis buffer containing protease inhibitors (Beyotime). The lysates were centrifuged at 15,000 g at 4°C for 5 min, and the supernatants were then collected. Protein concentrations were determined using a BCA protein assay (Beyotime). A total of 25 μg of protein from each sample was loaded and electrophoresed on an SDS-polyacrylamide gradient gel (SDS-PAGE), and the blots were then transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk at 37°C for 30 min. The membranes were incubated with anti-AANAT (1:1,000, ab3505, Abcam), anti-p-AANAT (phospho T29,1:1,000, ab3439, Abcam) and anti-GAPDH (1:1,000, CW0100, CWBIO) antibodies at 4°C overnight. The membranes were then rinsed with TBST and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, 1:1,000) at 37°C for 1 h. The blotted proteins were visualized using an ECL detection system (Thermo Scientific).