Invest. Ophthalmol. Vis. Sci ;48(1):1-10. doi: /iovs Figure Legend:

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From: The Mutant Human ND4 Subunit of Complex I Induces Optic Neuropathy in the Mouse Invest. Ophthalmol. Vis. Sci.. 2007;48(1):1-10. doi:10.1167/iovs.06-0789 Figure Legend: Allotopic ND4 map and immunoblot analysis. (A) Diagram showing the nuclear-encoded ND4 to which the ATPc targeting sequence was appended to the N terminus and the FLAG epitope appended to the C terminus. To generate the mutant R340H ND4 we exchanged the AGG encoding Arg for the CAC codon encoding His at codon 340. (B) Immunoblot of RGCs with anti-FLAG antibody. Mito-His: mitochondria isolated from RGCs transfected with the mutant human ND4 containing AAV shows a 52 kDa band consistent with expression of the mutant ND4FLAG fusion polypeptide with the 6-kDa targeting sequence cleaved off and a fainter unprocessed 58-kDa polypeptide retaining the targeting sequence. Mito-Arg: mitochondrial isolates from RGCs transfected with AAV expressing the normal human ND4 subunit gene show expression of the FLAG-tagged wild-type protein. Cytoplasmic isolates obtained from RGCs transfected with the AAV expressing the mutant human ND4FLAG (Cyto-His) or the wild-type ND4FLAG (Cyto-Arg) show no staining, with the anti-FLAG antibody indicating the absence of the fusion polypeptide in the cytoplasm. Retinal ganglion cells mock treated with AAV-GFP show the absence of FLAG in mitochondrial (Mito-Mock) and cytoplasmic (Cyto-Mock) isolates. For mitochondrial protein loading, the 31 kDa band of VDAC1/Porin is also shown. (C) A Coomassie blue-stained gel of complex I immunoprecipitated mitochondrial proteins from mutant ND4FLAG and mock-treated RGC-5 cells revealed multiple bands consistent with the different subunits of rat complex I (left), including a 52-kDa band. Immunoblot analysis with the anti-FLAG antibody revealed the 52-kDa band of mutant human R340H ND4 (middle) in cells transfected with mutant ND4FLAG (His) but not in cells mock treated with AAV-GFP (mock). FLAG immunoblot of complex I immunoprecipitated mitochondrial samples isolated from 20 FLAG tagged mutant human ND4 inoculated eyes revealed a strong signal at 52 kDa in the retina, but not in the optic nerve (right). (D) Transmission electron microscopy of mitochondria (boxed) of an RGC-5 cell transfected with AAV expressing human ND4FLAG revealed FLAG labeled immunogold (arrows) decorated the interior of mitochondria (E). The ND4FLAG immunogold is shown, at high magnification, inside the mitochondria of another RGC-5 cell (F). Date of download: 10/9/2017 The Association for Research in Vision and Ophthalmology Copyright © 2017. All rights reserved.