-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.