Supersignal west pico chemilunminescent substrate

Tissues or cells were homogenized in a non-denaturing lysis buffer that contained 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, with freshly added protease inhibitors tablet (Roche), and phosphatase inhibitor 2 mM sodium vanadate and 5mM sodium fluoride. The extract was centrifuged at 9500g for 10 min at 4 °C. Supernatants were collected and analyzed with BCA (Thermo Scientific) to quantify protein content. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Bio-Rad). Individual proteins were detected with specific antibodies and visualized on film using horseradish peroxidase-conjugated secondary antibodies (Fisher Scientific) and SuperSignal West Pico Chemilunminescent Substrate (Thermo Scientific). Bands were quantified with ImageJ (NIH).

Immunoprecipated MnSOD complex was resolved by SDS–PAGE and transferred to nitrocellulose membranes (Bio-Rad). nitrocellulose membranes were sequentially blocked by 5% milk and Avidin/Biotin blocking buffer. Biotinlyzed-E06 antibody was used to detect OxPL modification on MnSOD. E06 signal was visualized on film using horseradish peroxidase-conjugated NeutrAvidin (Life Technologies) and SuperSignal West Pico Chemilunminescent Substrate (Thermo Scientific).