Quantifying Retinal Protein Expression in Mice

All Western blot quantifications used 4 biological samples of 2-month-old mice with each sample consisting of both retinas from the same mouse. The analysis of each sample was performed in duplicate. Protein sample preparation and Western blot analysis were performed with the same reagents and techniques as previously described [14 (link)]. In brief, enucleated eyes were dissected in cold PBS buffer. Dissected retinas were immediately transferred into RIPA buffer (Thermo Scientific, cat# 89900) with protease and phosphatase inhibitors (1:100 dilution; cat#1861281) and homogenized by sonication. After 10 min centrifugation at 4 °C at 13,000 RPM, protein extracts were transferred into a fresh tube and protein concentration was quantified with the Bio-Rad Protein Assay (cat# 500-0113, 0114, 0115). To quantify PKM2 and p-S6 expression levels, 5 μg and 10 μg of total protein, respectively, were loaded. The following primary antibodies from Cell Signaling Technology were used: rabbit anti-PKM2 antibody (1:4000; Cat#4053), rabbit anti-pS6 (Ser240/244) (1:1000; Cat#5364), and for normalization, mouse anti-β-actin antibody (1:1000; Cat#3700). Protein detection was done using fluorescently labeled secondary (1:10,000) antibodies from Licor in combination with the Odyssey system. Quantification was performed with Image Studio software.