Oxidative stress-induced parkin sulfonation observed in vivo. (A) MPP+-induced ROS generation detected by the fluorogenic probe DCF in parkin-overexpressing SH-SY5Y cells. Catalase attenuated ROS production in cells exposed to MPP+. (B) ROS production induced by rotenone in a cell-based model of PD in primary striatal neurons. After exposure to 100 nM rotenone for 4 hours, 1 μM HEt was added for 30 min to assess ROS. Then the cells were fixed and immunostained for specific neuronal markers (both MAP2 and NeuN, purple), dopaminergic cells (TH, green), and nuclear DNA (Hoechst, blue). Scale bar, 10 μm. (C) Quantification of ROS intensity by deconvolution microscopy. Rotenone exposure led to ROS production and administration of catalase prior to rotenone reduced ROS generation. Data are expressed as mean ± SEM, n = 3; *p < 0.05 against Control; #p < 0.05 for Rotenone vs. Rotenone + Catalase by post-hoc ANOVA. (D) Parkin sulfonation in SH-SY5Y cells exposed to MPP+. Cell lysates were subjected to immunoprecipitation with anti-myc antibody (myc-IP). Exposure of parkin-overexpressing SH-SY5Y cells to 200 μM MPP+ for 18 hours resulted in a significant increase in parkin sulfonation in the "Insoluble" fraction. Administration of catalase 1 hour prior to MPP+ exposure prevented parkin sulfonation. Additional bands when probing for sulfonated parkin appeared only in the "Insoluble" fraction. (E) Parkin sulfonation was quantified by normalizing the intensity of sulfonated parkin to total parkin. Data are expressed as mean ± SEM, n = 4; *p < 0.01 for MPP+ vs. Control; #p < 0.01 for MPP+ vs. MPP+ + Catalase by post-hoc ANOVA.