To analyze the level of SNO-Drp1, the biotin-switch assay was performed as described previously (40). Briefly, the adult fly thoraxes were dissected and homogenized in HEN buffer [250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine]. Then, the free thiols in the tissue extracts were blocked with 20 mM methyl methanethiosulfonate (64306, Sigma) for 20 min at 50°C, and tissue extracts were precipitated with acetone. The pellets were harvested and resuspended in HEN buffer with 1% SDS. SNO was selectively reduced by ascorbate to reform the thiol group and subsequently biotinylated with N-[6-(biotinamido)hexyl]-3′-(2′pyridyldithio)-propionamide] (A35390, Thermo Fisher Scientific). The biotinylated proteins were pulled down with streptavidin-agarose beads (S1638, Sigma). Drp1 was detected by Western blotting with anti-Drp1 antibody (8570S, Cell Signaling Technology). Drp1-3HA and Drp1C643A-3HA were detected by Western blotting with anti-HA antibody (3724S, Cell Signaling Technology). Relative levels of S-nitrosylated proteins were determined by a comparison of S-nitrosylated Drp1 to the total amount of Drp1 in the extract for each sample.

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