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. 2013 Jul 16;3:2202. doi: 10.1038/srep02202

Figure 6. Peroxynitrite negatively regulates mitochondrial degradation.

Figure 6

(a) and (b) SH-SY5Y cells overexpressing parkin were treated with BAPTA-AM (10 μM) (a) or FeTPPS (5 μM) (b) with CCCP (10 μM) for 3 hr, and lysates were analysed by SNO-RAC. The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 3); *p < 0.05 and **p < 0.01 versus control respectively. (c) SH-SY5Y cells overexpressing wild-type or C323S parkin were incubated with CCCP (10 μM) for 6 hr and the peroxynitrite was measured by flow cytometry using APF (10 μM). Increases in peroxynitrite by CCCP were calculated as relative fold-changes in fluorescence compared to the untreated control. Data shown are mean ± SE (n = 4); *p < 0.05 versus wild type. (d) and (e) SH-SY5Y cells overexpressing wild-type or C323S parkin were incubated with CCCP (10 μM) for 3 hr. Cell lysates were immunoblotted with anti-nitrotyrosine and anti-beta-actin antibodies (d), or immunoprecipitated with anti-FLAG antibody and the immunoprecipitates were immunoblotted with anti-nitrotyrosine and anti-FLAG antibodies (e). The quantity of nitrotyrosine, as measured by scanning densitometry, is expressed as a percentage of CCCP-untreated wild-type, normalized with respect to beta-actin (d) or FLAG (e). Data shown are mean ± SE (n = 3); *p < 0.05, **p < 0.01 and N.S. represent significant and no significant difference versus the wild-type, respectively. # means p < 0.01 versus CCCP-untreated. (f) After preincubation with FeTPPS (5 μM), SH-SY5Y cells were treated with CCCP (10 μM) and FeTPPS (5 μM) for 3 hr, and then lysates were immunoblotted with anti-Tom20 and anti-beta-actin antibodies. Decreases in the level of Tom20 by CCCP were calculated as relative fold-changes in signal compared to the CCCP-untreated control, normalized with respect to beta-actin. Data shown are mean ± SE (n = 8); *p < 0.05 versus FeTPPS-untreated. Full scans of the blots in a, b, d and e are available in Supplementary Information, Fig.S10.