Western blotting

Retinas were homogenized in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and protease inhibitors). Homogenates were centrifuged for 20 min at 14.000 G, 4 °C to remove insoluble material. Protein concentration was determined by the BCA method (Thermo Scientific, Rockford, IL, USA, catalog # 23225) and bovine serum albumin was used as the standard, following manufacturer protocol. Total protein was separated in a 10% polyacrylamide electrophoresis gel and transferred to nitrocellulose membranes. Blots were blocked with 5% non-fat milk in the TBST buffer for 2 h at room temperature. After rinsing with TBST, blots were incubated overnight with primary antibodies in TBST/3% non-fat milk, according to Table ​Table1.1. After incubating with primary antibodies, blots were rinsed with TBST and incubated with goat anti-peroxidase (ECL^TM kit; Amersham, Buckinghamshire, England) for 2 h at room temperature. Detection of labeled proteins was achieved by using the enhanced chemiluminescent system (ECL^TM kit; Amersham). Measurements of optical densities (O.D.) of the bands were performed using ImageJ software (National Institute of Mental Health, Bethesda, Maryland, USA). O.D. values were normalized using the value obtained in the control group. L26 protein or amido black total protein staining were used as intern control, according to the indication in the respective figure legend. The SOD1/VDAC1 O.D. correlation was calculated using Pearson’s coefficient.

Antibodies used in this study.