Anti β actin

All proteins were extracted using a radio immunoprecipitation assay (RIPA) lysis buffer with protease and phosphatase inhibitors. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk solution for 1 h at room temperature. The membranes were then probed with anti-PPARγ (sc-7273, 1:200, Santa), anti-mTOR (#2983, 1:1000; Cell Signaling Technology), p-mTOR (#5536, 1:1000; Cell Signaling Technology), anti-ULK1 (#8054, 1:1000; Cell Signaling Technology), anti-LC3II (ab48394, 1:2000, Abcam), anti-P62 (ab56416, 1:2000; Abcam) and anti-β-actin (1:2000; Zhongshan Golden Bridge Biotechnology) overnight at 4 °C. The following day, membranes were washed three times with TBST and incubated with goat anti-rabbit or goat anti-mouse antibody IgG (H + L)-HRP (1:5000; Wuhan Sanying) for 1 h at room temperature. Finally, the membranes were visualized using the enhanced chemiluminescence substrate LumiGLO (Millipore, MA, USA).

Brain tissues were homogenized in the RIPA-DOC buffer supplemented with PMSF and phosphatase inhibitor (Roche). The lysed samples were centrifuged at 12,000 for 30 min at 4°C. The supernatants were used for Western blot analysis. The lysates were resolved on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane at 4°C. The membranes were blocked with 5% skim milk in TBST for 4 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-3 (3:5,000, Wanlei), anti-GRP78 (1:1,000; Cell Signaling), anti-CHOP (3:5,000; Wanlei), and anti-β-actin (1:2,000; Zhongshan Golden Bridge Biotechnology). Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (1:5,000, Zhongshan Golden Bridge Biotechnology) for 1.5 h at room temperature or 3 h at 4°C, followed by chemiluminescence assay. The optical density was quantified by Image-Pro Plus VERSion 6.0 (Media Cybernetics Inc., Bethesda, MD, USA). The experiments were not performed in a blinded manner.

Total retinal tissue lysates (n=6) were prepared by the addition of 500 µl sodium dodecyl sulfate (SDS) buffer [250 mM Tris (pH 6.8), 10% SDS, 500 mM dithiothreitol, 50% glycerol, 0.5% bromophenol blue and a 1:100 protease inhibitor cocktail (cat. no. P8340; Sigma-Aldrich)]. Total tissue extracts (20 µg) were separated on a 12% SDS polyacrylamide gel. Proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA) and blocked with 5% skim milk in TBST (TBS containing 0.05% Tween-20) at room temperature for 2 h. Subsequently, the membranes were hybridized at 4°C overnight in TBST with the primary antibodies: Anti-rPARP-1 (1:5,000; cat. no. 1051-1; Epitomics, Burlingame, CA, USA), and anti-rCaspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Boston, MA, USA) overnight at 4°C. Following incubation with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody, proteins were visualized with an enhanced chemiluminescence kit (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA). For normalization, blots were probed with β-actin, a housekeeping antibody (anti-β-actin; 1:1,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). The signal was detected by exposing X-ray films to the processed blots and analyzed by laser scanning densitometry (Personal Densitometer; GE Healthcare, Piscataway, NJ, USA).