Figure 1

H ₂ O ₂ -induced cysteine protein sulfonation observed in vitro. (A) Dot blot analysis of anti-sulfonation antibody to show dose-response to BSA sulfonation. (B and C) Immunoblot detection with anti-sulfonation pAb of sulfonated purified BSA or PTP1B after oxidative challenge with H₂O₂. BSA or PTP1B (10 μM) was exposed to 20 or 200 μM H₂O₂ for 30 min. For the competition assay, the anti-sulfonation pAb was incubated with 1 mM cysteic acid or cysteine sulfinic acid at RT for 2 hours prior to the incubation with the membrane. (D) Quantification of the competition assays for PTP1B sulfonation. Sulfonation competition was analyzed by normalizing to the intensity of PTP1B sulfonation (PTP- SO₃H) without H₂O₂ exposure. Data are expressed as mean ± SEM, n = 3; *p < 0.05 vs. PTP- SO₃H by post-hoc ANOVA. (E) Detection of sulfonated Prx from cells under oxidative stress induced by H₂O₂. Neuroblastoma SH-SY5Y cells were exposed to H₂O₂, followed by immunoblotting with antibodies against per-sulfonyl-BSA, Prx-SO₃ and Prx, respectively. Both anti-sulfonation antibodies detected a similar level of sulfonated Prx (Prx-SO₃H). (F) Parkin sulfonation after oxidative stress in vitro. Recombinant parkin was exposed to 20 or 200 μM H₂O₂ for 30 min, resulting in sulfonation by immunoblot analysis.