The effect of H₂O₂ on overoxidation of Jurkat and erythrocyte Prx2. (A) Jurkat cells were treated with the indicated H₂O₂ concentrations and immunoblotted with antibodies against overoxidized Prx (Prx-SO₂H). Protein (25 μg) was loaded per lane. (B) Erythrocytes (5 × 10⁶/mL) were pretreated with 1 mM azide for 5 minutes where specified, then treated with the indicated H₂O₂ concentrations for 10 minutes. Immunoblotting was then performed under nonreducing conditions against Prx-SO₂H (top panel), and under reducing conditions against Prx2 to serve as a loading control (bottom panel). J indicates 40 μg extract from Jurkat cells treated with 200 μM H₂O₂ for 10 minutes as in panel A as a positive control. The nonspecific band was consistently present in untreated Jurkat extracts. NS indicates nonspecific band; D, overoxidized Prx dimer; and M, overoxidized Prx monomer.