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

Figure 1. S-nitrosylation of parkin enhances the E3 ligase activity and mitochondrial degradation.

Figure 1

SH-SY5Y cells transfected with the FLAG-tagged wild-type parkin plasmid were treated with 1 μM rotenone (a) or 10 μM CCCP (b) for the indicated times, and cell lysates were analysed by SNO-RAC, in which ascorbate-dependent purification demonstrates the presence of S-nitrosylated cysteine residues, followed by immunoblot analysis with anti-parkin antibody (upper panels). 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 are means ± SE (n = 4); **p < 0.01 versus control (lower panels). (c) SH-SY5Y cells were transfected with the FLAG-tagged parkin plasmid and treated with GSNO (50 μM) for the indicated times. Lysates were used for immunoprecipitation with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-ubiquitin (upper panel) or anti-FLAG (middle panel) antibody and lysates were immunoblotted with anti-ubiquitin as control (lower panel). (d) Lysates prepared from HeLa cells treated with 50 μM GSNO and/or 10 μM CCCP for different time periods as indicated were immunoblotted with anti-Tom20, anti-HSP60 or anti-beta-actin antibodies (upper panel). The quantity of Tom20 and HSP60, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to beta-actin (lower panel). Data shown are mean ± SE (n = 4); **p < 0.01 versus GSNO-untreated samples, and # means p < 0.01 versus control. (e) HeLa cells transfected with the Venus-tagged parkin expression plasmid were incubated with 50 μM GSNO for 3 hr, and then immunostained with anti-Tom20 and anti-HSP60 antibodies (upper panel). The dotted line surrounded a parkin-expressing cell. The compaction index was calculated from the images stained with anti-Tom20 antibody as described in Materials and Methods (lower panel). Data shown are mean ± SE (n = 15); **p < 0.01 versus GSNO-untreated samples. Scale bars in images = 10 μm. Full scans of the blots in a, b and d are available in Supplementary Information, Fig.S10.