Chemidoc xrst image system

Cells were lysed using RIPA buffer in the presence of proteinase inhibitor cocktail and phosphatase inhibitor (Santa Cruz Biotechnology, Dallas, TX). Protein concentration was determined by a BCA Protein Assay (Thermo Scientific) using a FLUOstar OPTIMA microplate reader (BMG). Equal amounts of proteins were loaded and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, followed by Western blot analysis using standard protocols. The primary antibodies were: anti-NRF2 antibody (Millipore), Anti-KEAP1 antibody (Proteintech), anti-cleaved caspase 3 and caspase 3 antibodies (Cell Signaling), anti-cleaved PARP and PARP antibodies (Cell Signaling), anti-histone H3 antibody, anti-β-tubulin antibody (Cell Signaling), and anti-β-actin antibody (Sigma). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology. Immunoreactive protein bands were visualized by enhanced chemiluminescence and images were captured by a ChemiDoc XRST Image System (Bio-Rad).

CPB cells were treated with ABS (pH4, 200 μM) for 20 min, then cells were cultured with regular medium with 5 μM Dimedone for 1 h. Cells were harvested and co-immunoprecipitation (co-IP) was performed with antibody against KEAP1 (Proteintech, 10503–2-AP, Rosemont, IL) using Dynabeads protein A for immunoprecipitation (Thermofisher). The input and IP proteins were loaded and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. We used a rabbit antibody which recognizes all forms of cysteine with an oxidized thiol group (sulfenic RSOH, sulfinic RSO2H and sulfonic RSO3H) (Enzo Life Science, Farmingdale, NY)[22 (link)]. To avoid heavy chain background, a second mouse antibody against rabbit IgG light chain was applied (Cell Signaling, Danvers, MA), followed by anti-mouse IgG with HRP (Cell Signaling). Immuno-reactive protein bands were visualized by enhanced chemiluminescence (Thermofisher) and images were captured by a ChemiDoc XRST Image System (Bio-Rad).