*2.4. Western Blotting*

Retinal proteins were extracted through sonication in 80 μL of lysis buffer (phenylmethanesulfonylfluoride (PMSF), 1 mM; NaF, 100 mM; Na3VO4, 2 mM; all diluted in RIPA buffer (Sigma, St Louis, MO, USA)) and containing 1X protease inhibitor cocktail (Sigma, St Louis, MO, USA). Twenty-five micrograms protein of each sample were loaded in a 10% (*w*/*v*) SDS-PAGE, and electrophoresis was assessed at 90 V and 120 V for 30 and 60 min, respectively. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Madrid, Spain) at 400 mA for 90 min at 4 ◦C and blocked in 5% skimmed milk powder (Central Lechera

Asturiana, Siero, Spain) in 0.1% TBS-Tween. Primary antibodies (Table 1) were incubated at 4 ◦C overnight. Secondary antibodies goa<sup>t</sup> anti-rabbit and goa<sup>t</sup> anti-mouse (Dako Agilent, Santa Clara, CA, USA) were diluted 1:10,000 and the following day they were applied for 1 h at room temperature. Immunoreactive bands were detected using WesternBright ECL kit (WesternBright ECL HRP substrate, K-12045-D50, Advansta, CA, USA). Anti-vinculin (1:7000, sc-73,614; Santa Cruz, Dallas, TX, USA) and anti-cyclophilin A (1:10,000; BML-SA296; Enzo, NY, USA) were used to normalize protein levels. The densitometric analysis was carried out with Image J software (National Institutes of Health, Bethesda, MD, USA).


**Table 1.** Primary antibodies, targets, specific dilutions and sources used in western blot analysis.
