NF-κB western blotting

Mice were sacrificed by cervical dislocation and eyeballs were enucleated. Twelve retinas out of 30 retinas per experimental group, were used for western blot analyses; in particular, retinas were dissected from each eye, then two retinas from different mice of the same experimental group were pooled in a vial and homogenized (n = 6 independent retinal samples per experimental group). To assess the activation of the inflammatory pathway in the retina of DBA/2J mice, we analysed the phosphorylation of nuclear factor kappa-B (NF-κB) by western blotting (pNF-κB p65). Eyes were harvested, and retina collected and then homogenized in RIPA buffer containing a protease and phosphatase inhibitors cocktail (Sigma-Aldrich, St. Louis, MO). The protein concentrations of retinal homogenates were quantified with the BCA Assay Kit (Pierce™ BCA Protein Assay Kit, Invitrogen, Life Technologies, Carlsbad, USA). Equal amounts of protein samples (40 μg) were added to the Laemmli buffer (Bio-Rad), boiled at 95 °C for 5 min, loaded on 4–15% Mini-Protean TGX precast gels (Bio-Rad), then subjected to electrophoresis. The separated proteins were transferred onto a 0.2-μm polyvinylidene difluoride membranes (PVDF, Bio-Rad). Membranes were blocked in 5% milk in 1× Tris-buffered saline + Tween (TBST) for 1 h at room temperature. The blocked membranes were incubated with primary antibodies for phospho-NF-κB p65 (Ser536; mouse mAb #3036 Cell Signaling Technology, MA, USA, 1:1000), NF-κB p65 (XP® Rabbit mAb #8242 Cell Signaling Technology, MA, USA, 1:1000), and GAPDH (AB2302 Millipore, Burlington, MA, USA, 1:2000), overnight at 4 °C. Membranes were washed three times for 15 min and then incubated with secondary antibodies (1:10000, ECL anti-mouse, NA931; ECL anti-rabbit NA934, GE Healthcare, IL, USA) for 1 h at room temperature. Membranes were washed three times for 15 min and enhanced chemiluminescence (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, Carlsbad, CA, USA) solution was used for immunodetection. The relative density of the protein bands was normalized to the levels of GAPDH. The immunoblot bands intensities were quantified using ImageJ software for gel densitometry (provided in the public domain by the National Institutes of Health, available online: http://rsbweb.nih.gov/ij/download.html)