The human pathogenic NDUFV2 deletion mutant lost most of its mitochondrial targeting ability. (a) Schematic representation of the genomic structure of NDUFV2 in the first three exons. The 4-bp deletion of human pathogenic IVS2+5_+8delGTAA mutation is indicated by underlined red letters (GTAA). Dotted line represents the wild-type splicing form, and continuous line indicates the abnormal splicing form. Schematic structures of c-myc fusion proteins corresponding to the wild-type splicing form and the abnormal splicing form are also shown here. E1, E2 and E3 represent exon 1, exon 2 and exon 3, respectively. (b) The protein distribution patterns of wild-type and NDUFV2 IVS2+5_+8delGTAA mutant. The expressed proteins were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. Scale bars = 10 μm. (c) Subcellular localization of the wild-type and NDUFV2 pathogenic mutant. Western blot analyses were conducted using cytosolic (Cy) and mitochondrial (Mi) extracts from T-REx-293 cells transiently transfected with the wild-type or NDUFV2 pathogenic mutant construct. (c) The expression levels of the wild-type and the △19-40 NDUFV2 mutant proteins in the whole cell lysates. Western blot analyses were conducted using the whole cell lysates from T-REx-293 cells transiently transfected with the wild-type or the △19-40 NDUFV2 mutant construct. The expressed proteins were detected by an anti-c-myc antibody. β-tubulin was used as a cytosolic marker and ATP synthase subunit α (ATP α) was used as a mitochondrial marker.