In contrast, the T. brucei TRF protein

(TbTRF) appears to co-localize with most telomeres at all stages of the cell cycle in both bloodstream and procyclic forms [24]. Whether LaTRF also has other cellular roles or if its association with telomeres occurs in a cell cycle dependent manner is not clear at this stage. Figure 3 LaTRF partially co-localizes with L. amazonensis telomeres. LaTRF (red), using anti-LaTRF serum, was combined with FISH (green) using a PNA-telomere probe specific for TTAGGG repeats. DAPI (blue) was used to stain DNA in the nucleus (N) and in the kinetoplast see more (K). Images were organized in panels p1-p4 showing the co-localization patterns in merged (a): telomeres and LaTRF, and in merged (b): DAPI, telomeres and LaTRF. Merged images were done using NIS elements software (v. Br 2.30). LaTRF interacts in vitro and in vivo with L. amazonensis telomeres using a Myb-like DNA binding domain EMSA assays were done with renatured protein extracts containing full length LaTRF, the Myb-like DNA binding domain (LaTRFMyb) (Figs 4 and 5, see Selleckchem WZB117 additional file 1) and with L. amazonensis nuclear extracts (Fig 6), to investigate whether LaTRF, like its vertebrate and trypanosome counterparts [18, 24], was able to bind double-stranded telomeric DNA in vitro. Figure 4 Recombinant LaTRF and the mutant bearing

the C-terminal Myb domain bind in vitro double-stranded telomeric DNA. Electrophoretic selleck kinase inhibitor mobility shift assays (EMSA) were done using radiolabeled double-stranded telomeric DNA (LaTEL) as probe. Protein:DNA complexes were separated in a 4% PAGE in 1X TBE. EMSA was done with E. coli BL21 protein extract (lane 2), recombinant full length LaTRF (lanes 3-6) and a mutant bearing the C-terminal Myb domain (lanes 7-9). A supershift assay many was done with anti-LaTRF serum (lane 6). Assays were also done in the presence of 20 fold excess of non-labeled LaTEL as specific competitor (lanes 4 and 8) or 100 fold excess of double-stranded non-specific poly [dI-dC] [dI-dC] DNA (lanes 5 and 9). In lane 1, no protein was

added to the binding reaction. The original gel image and its content are shown as additional file 1: Figure S1. Figure 5 Supershift and competition assays confirm that recombinant full length LaTRF bind in vitro double-stranded telomeric DNA. Electrophoretic mobility shift assays (EMSA) were done using radiolabeled double-stranded telomeric DNA (LaTEL) as probe. Protein:DNA complexes were separated in a 4% PAGE in 1X TBE. EMSA was done with recombinant full length LaTRF and anti-LaTRF serum in the absence (lane 2) and in the presence of 20 fold excess of non-labeled LaTEL as specific competitor (lane 3) or 100 fold excess of double-stranded non-specific DNA (poly [dI-dC] [dI-dC]) as non specific competitor (lane 4).