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A log phase culture of A-549 cells was transfected with an pEYFP-Mitochondria plasmid subcellular localization vector, which contains the mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase. The enhanced yellow fluorescent protein gene employed with this culture features several important amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by approximately 18 nanometers, from 509 to 527 nanometers. The cells were additionally labeled with Alexa Fluor 568 conjugated to phalloidin and DAPI, targeting the filamentous actin network and nuclei, respectively.
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