8 × 108 cells/experiment) as described by Lira et al. [17]. Chromatin was immunoprecipitated selleck screening library with anti-LaTRF serum and DNA was extracted after cross-link reversal. DNA samples were slot-blotted and hybridized with Tel1 and kDNA probes by using a previously established protocol. Aliquots of 1% and 10% of total DNA used in each experiment (input) were tested separately. Control assays included immunoprecipitation of chromatin with pre-immuneserum (pre-immune) or without serum (mock). The probes used were 5′-end labeled with γATP [32P]: Tel1 (5′TTAGGG-3′)3 and kDNA (5′-TTTCGGCTCGGGCGGTGAAAACTGGGGGTTGGTGTAAAAT-3′), according to Lira et al. [17]. Acknowledgements The authors thank Drs. S.

Hyslop and J.P. Monteiro for revising the English version of the manuscript. This work was supported by FAPESP (06/58175-7) and CNPq (481850/2008). MSS is supported by an undergraduate

studentship from FAPESP. AMP is supported by a doctoral studentship from FAPESP. RCVS and CEM are respectively CHIR-99021 in vitro supported by doctoral and master studentships from CNPq (Brazil). Electronic supplementary material Additional file 1: Figure S1. Original and unmanipulated gel image shown in figure 4. EMSA done with radiolabeled double-stranded telomeric DNA (LaTEL) as probe. Protein:DNA complexes were separated in a 4% PAGE in 1X TBE. In lane 1, no STI571 research buy protein was added to the binding reaction. In lane 2, EMSA was done with E. coli BL21 protein extract. In lane 3, EMSA was done with recombinant full length LaTRF. In lane 4, EMSA was done with recombinant full triclocarban length LaTRF in the presence of 20 fold excess of non-labeled LaTEL as specific competitor. In lane 5, no protein was added to the binding reaction (as in lane 1). In lane 6, EMSA was done with recombinant full length LaTRF in the presence of 100 fold excess of double-stranded non-specific poly [dI-dC] [dI-dC] DNA. In lane 7, EMSA was done with recombinant full length

LaTRF in the presence of anti-LaTRF serum (supershift assay). Please check the supershifted complex at the top of the lane. In lane 8, EMSA was done with the mutant recombinant protein bearing the C-terminal Myb domain. In lane 9, EMSA was done with the mutant recombinant protein bearing the C-terminal Myb domain in the presence of 20 fold excess of non-labeled LaTEL. In lane 10, the same experiment shown in lane 9. In lane 11, EMSA was done with the mutant recombinant protein bearing the C-terminal Myb domain in the presence of 100 fold excess of double-stranded non-specific poly [dI-dC] [dI-dC] DNA. In lane 12, the same supershift assay shown in lane 7. (PNG 466 KB) Additional file 2: Table S1 Primers used for PCR amplification and sequencing of the putative L. amazonensis TRF gene and the deletion mutant LaTRFMyb. Table containing a list of the primers used for PCR and sequencing assays (DOC 30 KB) References 1.