Western Blot Analysis of Retinal Proteins

Western blot was performed on proteins extracted from a single retina [16 (link)]. Between seven and sixteen individuals were tested per condition. Harvested retinas were snap frozen before lysis in 80 μL P300 buffer (20 mM Na₂HPO₄; 250 mM NaCl; 30 mM NaPPi; 0.1% Nonidet P-40; 5 mM EDTA; 5 mM DTT) and protease inhibitor cocktail (Sigma-Aldrich, Saint-Quentin Fallavier, France). Protein content was measured using the Lowry protein assay kit (DC Protein Assay; Bio-Rad, Marnes-la-Coquette, France). Homogenates were sonicated and centrifuged for 15 min at 5 000 g, and then 10 μg of the supernatant were subjected to SDS-PAGE, as previously described [12 (link)]. Western blots were then conducted using standard procedures. Primary and secondary antibodies are listed in Supplementary Table S3. Antibody binding was revealed by the Enhanced Chemiluminescence System (Bio-Rad) on X-Ray film (Sigma-Aldrich). Each sample was probed once with anti-α-tubulin antibody for normalization. Quantification was done using ImageJ software [71 (link)]. All original western blot gels are depicted in Supplementary Fig. 4.