Horseradish peroxide conjugated anti mouse and anti rabbit antibodies

At the time of sacrifice, left eye was washed, collected, and lysed using protein extraction solution (iNtRON, Seoul, Korea). The obtained protein was quantified using a BCA protein assay kit (BIO-RAD, Hercules, CA, USA). The subsequent procedure was used slightly modified from the experimental method of the previous study [19 (link)]. Primary antibodies against caspase-3, Nrf2, and HO-1 were purchased from Santa Cruz Biotechnology (CA, USA) and antibody against β-actin was obtained from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies were horseradish-peroxide-conjugated anti-mouse and anti-rabbit antibodies (Cell Signaling Technology). The protein bands were developed by ATTD Corporation (Tokyo, Japan).

ARPE-19 cells were lysed using radioimmunoprecipitation (RIPA) buffer (ThermoFisher Scientific, Waltham, MA, USA) for 1 h on ice. After centrifugation at 10,000 rpm for 10 min at 4 °C, whole-cell lysates were collected. Equal amounts of protein (40 μg), measured by standard curves using bovine serum albumin (BSA, Sigma-Aldrich), were boiled for 5 min. The proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked with TBS-T buffer (0.1% Tween-20 containing Tris-buffered saline (TBS, Bio-Rad)) containing 5% skim milk and then incubated with primary antibodies overnight at 4 °C. Primary antibodies against caspase-3/-8/-9, PARP, Nrf2, HO-1, p21, cdk2, and cyclin A were obtained from Santa Cruz Biotechnology and antibody against β-actin was purchased from Cell Signaling Technology. The membranes were washed in TBS-T buffer five times and then incubated with secondary antibodies for 2 h at room temperature. The secondary antibodies were horseradish-peroxide-conjugated anti-mouse and anti-rabbit antibodies (Cell Signaling). The protein bands were developed and analyzed by a chemiluminescence imaging system provided by ATTD Corporation.