On day 7 after immunization, retinas of five EAU mice each of nude, WT, TLR4−/−, iNOS−/−, TNF-α−/−, five WT mice injected with pertussis toxin and CFA alone, and five nonimmunized WT (control) mice were dissected, homogenized, and lysed in protein extraction buffer (M-PER; Thermo Scientific, Waltham, MA) containing protease inhibitors (Calbiochem, San Diego, CA). The homogenates were then sonicated for 30 seconds and centrifuged at 13,000 rpm for 20 minutes at 4°C. Protein quantification of the supernatant was determined using bovine serum albumin as the standard (Bio-Rad Laboratories). Equal amounts of protein samples were loaded and run on SDS-PAGE (15% Tris-HCl polyacrylamide ready gels [Bio-Rad Laboratories]; to detect iNOS, 7.5% Tris-HCl polyacrylamide ready gels were used (Bio-Rad Laboratories). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) using a transblot semidry system. The membranes were blocked using 5% skim milk and then probed with a polyclonal anti–αA-crystallin (Stressgen, Ann Arbor, MI) at a 1:2500 dilution overnight at 4°C. Similarly, iNOS and TNF-α were detected by probing the membranes with polyclonal anti-iNOS (BD Transduction Laboratories, San Jose, CA) and monoclonal TNF-α (Santa Cruz Biotechnology, Santa Cruz, CA), respectively, with iNOS at a 1:2000 dilution and TNF-α at a 1:500 dilution. After incubation for 45 minutes with the secondary antibody tagged with horseradish peroxidase (anti-mouse or anti-rabbit, depending on the primary antibody used; Santa Cruz Biotechnology), signals were detected by chemiluminescence system (Thermo Scientific). Equal protein loading of retinal lysates from each group of animals was confirmed by reprobing blots with a monoclonal antibody to β-actin.