Characterization of Tb11.NT.28. (A) Sequence conservation of Tb11.NT.28 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. The predicted transmembrane domain (TM helix) is indicated. (B) Immunofluorescence analysis of cells expressing a C-terminal HA-tagged Tb.11.NT.28. Cells were stained with an anti-HA antibody (green) and MitoTracker (red) and a merge of signal for HA and MitoTracker is shown in yellow. (C) Cell fractionation of cells expressing C-terminal HA-tagged Tb11.NT.28. Western blot analysis was performed against C-terminal HA-tagged Tb11.NT.28 (top panel), RNA-editing associated protein (REAP, middle panel) and cytoplasmic HSP70 (bottom panel) on total (T), cytoplasmic (C), nuclear (N), endoplasmic reticulum (ER) and mitochondrial (Mito) fractions. (D) Digitonin extraction analysis of cells expressing C-terminal HA-tagged Tb11.NT.28. Parasites were exposed to increasing concentrations of the detergent digitonin as indicated above the panels and solubilized proteins were analyzed by Western blot for HA-tagged Tb11.NT.28 (top panel), trypanosome alternative oxidase (TAO, middle panel) and mitochondrial HSP 70 (mtHSP70, bottom panel).
Ericson et al. BMC Biology 2014 12:14 doi:10.1186/1741-7007-12-14