Figure 6

S-nitrosylation impaired the E3 ubiquitin ligase activity of parkin and inhibited the ubiquitination of DMT1. (a) WT-parkin was co-transfected with Myc-DMT1 and HA-Ub into HEK293T cells for 24 h. The ubiquitination of DMT1 presented. With the GSNO treatment, the ubiquitination of DMT1 was inhibited. (b) Single site mutant (Cys241A, Cys260A, Cys323A)-parkin was co-transfected with Myc-DMT1 and HA-Ub into HEK293T cells for 24 h. With the GSNO treatment, the ubiquitination of DMT1 was inhibited also. (c) Mutants (Cys241A, Cys260A, Cys323A)-parkin simultaneously, were co-transfected with Myc-DMT1 and HA-Ub into HEK293T cells for 24 h. With the GSNO treatment, the ubiquitination of DMT1 was not inhibited. Panels were representative of 3 independent experiments.