Recombinant Aurora A (0.5 μg) was incubated with 50 mM tris-HCl (pH 7.4) and 100 mM NaCl in the presence of different concentrations of H2O2 or 10 mM DTT for 10 min. Cysteine sulfenic acid was detected by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotting after adduct formation with 1 mM dimedone for 20 min. Dimedone stocks were prepared in DMSO with a final assay DMSO concentration no higher than 4% (v/v). To detect glutathionylation of proteins, 1 μg of proteins was incubated with 10 mM GSSG or GSH for 30 min, and glutathione-protein complexes were detected by immunoblotting after nonreducing SDS-PAGE. Comparative changes in the electrophoretic mobility of Aurora A in the presence of DTT and H2O2 were also detected with antibodies for total and pThr288Aurora A, and SDS-PAGE was performed under reducing and nonreducing conditions. All incubations were performed at 20°C.

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