FIG. 3.

Analysis of the specificity of antibodies raised against N-terminal peptides of cyto-LysRS and mito-LysRS from human. (a) The yeast extracts described in Fig. 2 were analyzed by Western blotting using antibodies directed to full-length LysRS (IgG × KRS) or to synthetic peptides specific for the mitochondrial (IgG × mKRS) or cytoplasmic (IgG × cKRS) form of human LysRS. (b) Total extracts from HeLa or U937 cells were analyzed by Western blotting using antibodies directed to synthetic peptides specific for the mitochondrial (IgG × mKRS) or cytoplasmic (IgG × cKRS) form of human LysRS or antibodies directed to full-length LysRS (IgG × KRS). mKRS, polypeptide corresponding to mito-LysRS; x, a polypeptide cross-reacting with IgG × mKRS. (c) Total extracts of HeLa Tet-Off cells transfected with pTRE2hyg vectors that overexpressed the premitochondrial (pmKRS) or mitochondrial (mKRS) form of LysRS were analyzed by Western blotting using antibodies directed to the synthetic peptide specific for mito-LysRS (IgG × mKRS). The extracts were prepared 8 h, 24 h, or 32 h after transfection. C, a control extract of HeLa Tet-Off cells prepared before transfection. Note that the intensity of the cross-reacting polypeptide (x) is independent of transfection of HeLa cells with plasmids that express different forms of mito-LysRS. (d) A total extract of U937 cells (U937) or the cytoplasmic (cyto) or mitochondrial (mito) fraction obtained after subcellular fractionation of a U937 cell extract was analyzed by Western blotting using antibodies directed to full-length LysRS (KRS) or to cytochrome c (CytC) or AspRS (DRS), used as markers of the mitochondrial and cytoplasmic compartments, respectively. Lanes “cyto” and “mito” contain equivalent amounts of the initial extract of U937 cells. Lane “mito ×5” contains a fivefold excess of the “mito” fraction.