DCP-Bio1 labeling.

Cells were scraped and lysed, and they reacted with the cysteine sulfenic acid probe DCP-Bio1 in a modified lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% NP-40, 0.5% Triton X-100, 50 mM NaF, 1 mM PMSF, 1 mM DCP-Bio1, 100 μM diethylene triamine pentaacetic acid, 10 mM N-ethylmaleimide, 10 mM iodoacetamide, 200 U/mL CAT, and protease inhibitor cocktail. The cell lysates were incubated on ice for 1.5 hour and then centrifuged at 9,600g for 10 minutes at 4°C. Unreacted DCP-Bio1 in the supernatant was removed using Bio-Rad P6-Spin Columns following manufacturer’s instructions. General biotin-labeled oxidized proteins were visualized by Western blotting using Streptavidin-HRP. For identifying specific oxidized protein, the cell lysates were used for immunoprecipitation experiment.