Figure 3.

Subcellular localization of human HARS2 in 143B cells. A, cells were transiently transfected with a HARS2 cDNA fused with GFP. The fusion protein was visualized by indirect immunofluorescence using antibodies to GFP. Mitotracker Red–stained mitochondria and 4′,6-diamidino-2-phenylindole–stained nuclei were identified by red and blue fluorescence respectively. Scale bars, 50 μm. B, Western blot analysis of mitochondrial aminoacyl-tRNA synthetases in these four transfectants (C9V (vector only), C9H (exogenous HARS2), E1V (vector only), and E1H (exogenous HARS2)) and their parental cell lines E1 and C9. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with HARS2, LARS2, and SARS2, respectively, and with TOM20 as a loading control.