After permeabilization with 0.1% Triton X-100 (in 1X PBS) for 10 min at room temperature, cells were incubated with 0.1 M Glycine (in 1X PBS) and attached to glass coverslips coated with 0.1% poly-L-Lysine (Sigma). Anti-LaTRF serum was used selleck chemicals to detect LaTRF with Alexa Fluor 555-labeled goat anti-rabbit IgG (Invitrogen) as the secondary

antibody followed by telomere detection using a Telomere PNA FISH Kit/FITC (DakoCytomation). VECTASHIELD® Mounting Medium with DAPI (Vector Labs) was used as the anti-fade mounting solution and to stain nuclear and kinetoplast DNA. The images were analyzed with a Nikon 80i fluorescence microscope and captured with a digital camera (Nikon). When necessary, images were superimposed using NIS elements software (v. Br 2.30). EMSA (electrophoretic mobility shift assay) All of the conditions for binding reactions and EMSA, including binding temperature, PD173074 protein concentrations in the extracts and the double-stranded DNA probe (LaTEL), were standardized in preliminary experiments. LaTEL was constructed by using the γ [32P]ATP 5′-end-labeled oligonucleotides ssTel78G and ssTel78C, as described by Lira et al. [17]. Assays were done by mixing 10 μg of renatured bacterial extracts containing full length LaTRF or LaTRF Myb

with approximately 2 pmol of labeled probe (LaTEL) in 30 μl of EMSA buffer (20 mM HEPES, 2.5 Alvocidib ic50 mM MgCl2, 0.1 mM EDTA, 0.1 M KCl, 10% glycerol, 0.5 mM DTT, pH 8.0) containing 10 ng of poly [dI-dC] [dI-dC] and 10 ng of poly [dA-dT] [dA-dT]. Total protein extracts of non-transformed E. coli were used as controls. The reactions were incubated for 30 min at room temperature and loaded onto a non-denaturing 4% polyacrylamide gel (acrylamide:bis-acrylamide, 19:1, w/w) in 1X TBE. After electrophoresis, the gels were exposed to X-ray film. Binding reactions were also done with crude nuclear extracts obtained from 108 parasites

(~2.3 μg of total proteins) and pheromone γ [32P]ATP labeled LaTEL (2 pmol) in EMSA buffer containing a mixture of 10 ng of poly [dI-dC] [dI-dC] and 10 ng of poly [dA-dT] [dA-dT]. Competition assays to test the binding specificity of proteins in both recombinant and nuclear extracts, were done using 20 fold excess of unlabeled LaTEL (in relation to the labeled probe) as the specific competitor and a 100 fold excess (in relation to the labeled probe) of unlabeled double-stranded DNA poly [dI-dC] [dI-dC] as the non-specific competitor. Supershift assays were done using full-length recombinant LaTRF (10 μg) or native nuclear extracts from 108 parasites in the presence of ~30 μg of anti-LaTRF serum in EMSA buffer containing labeled LaTEL as probe and both poly [dI-dC] [dI-dC] and poly [dA-dT] [dA-dT] as above described. These assays were also performed in the presence of 20 fold excess of non-labeled LaTEL and 100 fold excess of poly [dI-dC] [dI-dC] as described above. Chromatin immunoprecipitation Formaldehyde cross-linked chromatin was obtained from promastigote forms of L.