FIGURE 3.

Endogenous nNOS interacted with full-length PINK1 accumulated during mitophagy. A, both PINK1-null and wild-type cells were treated with CCCP (10 μm for 24 h), and mitochondrial (Mito) lysates were analyzed by immunoblotting using an antibody against phospho-nNOS at Ser¹⁴¹² (left panel). Both PINK1-null and wild-type cell lysates were subjected to immunoblotting using anti-nNOS antibody (right panel). B, PINK1-null cells were transfected with either FLAG-tagged wild-type PINK1 or L347P mutant, and cell lysates were prepared. Phospho-nNOS accumulation was analyzed by immunoblotting using anti-phospho-nNOS antibody. Representative results are shown in A and B. C, wild-type cells were transfected with FLAG-tagged nNOS (green) and Su9-RFP (mitochondrial marker, red), followed by CCCP treatment. The colocalization of nNOS with mitochondrial Su9-RFP by CCCP was monitored by confocal microscopy. Representative images are shown. CON, control. D, HEK293T cells were transfected with myc-tagged nNOS and FLAG-tagged PINK1 plasmids as indicated. Immunoprecipitation was performed as indicated and as described under “Experimental Procedures.” IB, immunoblot. E, HEK293T cells were transfected with FLAG-tagged nNOS and myc-tagged PINK1 plasmids, and a reverse IP assay was performed as indicated. F, cell lysates of wild-type cells were prepared in the presence and absence of CCCP and incubated with nNOS or TOM 20 antibody and protein G-Sepharose overnight at 4 °C as indicated and as described under “Experimental Procedures.” A co-IP assay using either the endogenous nNOS and full-length PINK1 (left panel) or the endogenous nNOS and TOM20 (right panel) was performed. TOM20 was used as a negative control protein. G, HEK293T cells were transfected with myc-tagged nNOS and FLAG-tagged L347P mutant plasmids as indicated. An IP assay was performed as indicated. Representative images are shown in D--G. At least three independent experiments were performed.