Subcellular localization of JEV NS5.

<p>(A-C) Cellular lysates of HEK293T cells infected with JEV (MOI = 5) or transfected with NS5-Flag for 24 h underwent Qproteome Mitochondria Isolation (A) or biochemical fractionation as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004750#ppat.1004750.s005" target="_blank">S5B and S5C Fig</a>, respectively (B and C). Western blot analysis of indicated proteins in cytosolic and crude mitochondrial fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction; L, light microsomal membrane fraction. (D) The crude mitochondrial fraction isolated from HEK293T cells infected with JEV (MOI = 3) or transfected with NS5-Flag for 24 h was treated with Proteinase K (100 μg/ml) for 30 min on ice. The reactants were developed by Western blot analysis with antibodies against NS5 or the indicated mitochondrial proteins. (E) Confocal microscopy of pEYFP-Mito-NS5-A549 cells stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HADHα plus Alexa Fluor 647 goat anti-mouse antibody. (F) Confocal microscopy of pEYFP-Mito-NS5-A549 cells transfected with HADHβ-HA for 24 h and stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HA plus Alexa Fluor 647 goat anti-mouse antibody.