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. 2015 Oct 2;290(46):27644–27659. doi: 10.1074/jbc.M115.654129

FIGURE 4.

FIGURE 4.

Stable expression of plant NAD+ transporter AtNDT2 in 293 cells constitutively increases the mitochondrial NAD+ content. A, subcellular localization of AtNDT2 and human SCL25A32 in HeLa S3 cells transiently transfected with the vector for stable transfection. AtNDT2 and SLC25A32 are detected by their FLAG epitope, nuclei are stained with DAPI, and mitochondrial structures are stained with MitoTracker. Scale bar, 10 μm. B, fluorescence micrographs of stably transfected monoclonal 293 cells expressing C-terminally FLAG-tagged AtNDT2 (293AtNDT2) or SLC25A32 (293SLC25A32). AtNDT2 and SLC25A32 are detected by their FLAG epitope, and nuclei are stained with DAPI. All cells exhibit immunoreactivity toward the FLAG antibody. Scale bar, 10 μm. C, FLAG immunoblot analysis of 293AtNDT2 and 293SLC25A32 cell lysates. β-Tubulin served as a loading control. D, lysates from parental 293 cells and monoclonal 293AtNDT2 and 293SLC25A32 cells transiently transfected with the vector encoding mitoPARP (detected via its EGFP portion) were subjected to PAR immunoblot analyses. The level of PAR immunoreactivity reflects the mitochondrial NAD+ content. β-Tubulin served as a loading control.