Characterization of Tb10.NT.87. (A) Sequence conservation of Tb10.NT.87 across kinetoplastids. Amino acid sequences from T. brucei (Tbr), T. cruzi (Tcr) and L. major (Lma) were aligned with ClustalW and conserved residues are shaded with black boxes while similar residues are shaded in grey. (B) Live fluorescence microscopy of cells expressing a C-terminal GFP-tagged Tb10.NT.87. GFP-signal (green), MitoTracker (red), GFP and MitoTracker merge (yellow), Hoechst (blue) and DIC images are shown. (C) Immunogold electron microscopy of C-terminal HA-tagged Tb10.NT.87. Mitochondrial tubules indicated with the letter M and kinetoplast (mitochondrial DNA) indicated by an arrow and K. (D) Cellular fractionation of cells expressing C-terminal GFP-tagged Tb10.NT.87. Western blot against C-terminal GFP-tagged Tb10.NT.87 (top panel), editing protein (α-63/P1H3, middle panel) and cytoplasmic HSP70 (bottom panel) on total (T), cytoplasmic (C), nuclear (N), endoplasmic reticulum (ER) and mitochondria (Mito) fractions. (E) Digitonin extraction analysis of cells expressing C-terminal GFP-tagged Tb10.NT.87. Parasites were exposed to increasing concentrations of the detergent digitonin as indicated and solubilized proteins were analyzed by Western blot for GFP-tagged Tb10.NT.87 (top panel), mitochondrial HSP 70 (mtHSP70, middle panel), and trypanosome alternative oxidase (TAO, bottom panel). DIC, differential interference contrast.
Ericson et al. BMC Biology 2014 12:14 doi:10.1186/1741-7007-12-14