2 nrf2

The left eye was separated using protein extraction solution (iNtRON, Seoul, Korea), and the obtained protein was quantified using a BCA protein assay kit (BIO-RAD, Hercules, CA, USA). Equal amounts of protein were mixed 1:1 with sample buffer (Laemmli sample buffer (BIO-RAD) and 2-mercaptoethanol (BIO-RAD) mixed at 19:1) in each group and then heated at 99°C for 5 minutes. Heated protein was electrophoresed via 12% SDS polyacrylamide gel and transferred by electrophoretic means to nitrocellulose membrane. The membrane was blocked with 5% skim milk at room temperature, and then primary antibodies against the following proteins were used: rhodopsin (Abcam, Burlingame, CA, USA), opsin (Millipore, Burlington, MA, USA), heme oxygenase (HO-1, Cell Signaling, Beverly, MA, USA), nuclear enzyme erythroid 2-related factor-2 (NRF2, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (Cell Signaling), and poly (ADP-ribose) polymerase (PARP, Cell Signaling). Horseradish-peroxidase-conjugated anti-mouse, and anti-rabbit secondary antibody were diluted in TBS-T solution. All western blots were re-probed with anti-β-actin antibody (Cell Signaling) to ensure protein loading. Bands were visualized with EZ-capture ST (ATTD) reagents according to the manufacturer's instructions.

Colonic mucosal tissues were first lysed in RIPA buffer and subsequently homogenized on ice using a homogenizer. The supernatants of the homogenates were collected after centrifugation at 13500 × rpm at 4°C for 15 min. Total extracted proteins (50 μg) were separated by sodium-dodecyl sulfate gel electrophoresis, transferred to nitrocellulose membranes and blocked with 5% skim milk for 1 h at room temperature. Afterward, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were claudin-3 (1:800 dilution) (Abcam, Cambridge, United Kingdom), claudin-5 (1:800 dilution) (Abcam), occludin (1:400 dilution) (Abcam), zonula occludens-1 (ZO-1) (1:500 dilution) (Abcam), nuclear factor erythroid 2-related factor 2 (Nrf2) (1:500 dilution) (Santa Cruz Biotechnology, Dallas, TX, United States) and β-actin (1:8000 dilution). The membranes were washed with TBST buffer three times for 10 min each. Then, the membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (1:20000 dilution), after which chemiluminescence was detected. ImageJ software was used to measure the optical density of the bands. The expression levels of the target proteins relative to β-actin were calculated.