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

Figure 3. Effect of S-nitrosylation on the activity of parkin.

Figure 3

(a) SH-SY5Y cells were transfected with the wild-type or mutants parkin and treated with 10 μM MG132 for 3 hr. Lysates were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-ubiquitin or anti-FLAG antibody and lysates were with anti-ubiquitin as control as indicated. The quantity of ubiquitinated parkin, as measured by scanning densitometry, is expressed as a percentage of wild type treated with MG132, normalized with respect to total parkin and ubiquitinated proteins. Data shown are mean ± SE (n = 4); N.S. means no significant difference versus the wild-type. (b) E3 ligase activity of parkin was analysed in vitro using wild-type and C323S parkins purified from HEK293 cells. Immnoblot analyses were performed using anti-ubiquitin antibody (Ub-parkin) or anti-FLAG antibody (Total parkin). (c) SH-SY5Y cells were transfected with the FLAG-tagged wild-type or mutants parkin and treated with 50 μM GSNO for 3 hr in the presence of 10 μM MG132. Lysates were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-ubiquitin or anti-FLAG antibody, and lysates were with anti-ubiquitin as control as indicated. The quantity of ubiquitinated parkin, as measured by scanning densitometry, is expressed as a percentage of wild-type without GSNO treatment, normalized with respect to total parkin and ubiquitinated proteins. Data shown are mean ± SE (n = 4); **p < 0.01 and N.S. means no significant difference. (d) SH-SY5Y cells were transfected with the wild-type or C323S parkin and treated with 1 μM rotenone and 10 μM MG132 for 3 hr. Lysates were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-ubiquitin or anti-FLAG antibody, and lysates were with anti-ubiquitin as control as indicated. The quantity of ubiquitinated parkin, as measured by scanning densitometry, is expressed as a percentage of wild-type without rotenone treatment, normalized with respect to total parkin and ubiquitinated proteins. Data shown are mean ± SE (n = 4); **p < 0.01 and N.S. mean significant and no significant difference, respectively. Full scans of the blots in b are available in Supplementary Information, Fig.S10.