Western Blotting Analysis of Retinal Proteins

Western blotting analysis was performed as previously described [48 (link),49 (link)]. In brief, whole retinas were isolated and homogenized in ice-cold lysis buffer, supplemented with protease and phosphatase inhibitor cocktail (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein concentration was detected using a BCA assay kit (Beyotime). Equal amounts of protein (20 μg) were loaded in each lane, separated by 10% SDS-PAGE and electrically transferred to a polyvinylidene fluoride membrane. After blocking in 5% tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% skimmed milk, the membranes were incubated with primary antibodies for APP (1:10,000, ab32136, Abcam, Cambridge, MA, USA), Aβ₄₂ (1:10,000, ab2539, Abcam), phospho-tau S231 (1:3000, ab151559, Abcam), phospho-tau S396 (1:10,000, ab109390, Abcam) or β-actin (1:1000, 4970S, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4 °C. After incubation with the horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized by using a BeyoECL Plus kit (Beyotime). The intensity of bands was analyzed using ImageJ software (http://imagej.nih.gov/ij/).