All genes had the stop codon inserted in the reverse oligonucleotide, with exception of centrin that uses the stop codon of vector. The PCR products were then inserted into pDONR 221 (Invitrogen) by BP recombination and then transferred to pTcGW vectors by LR recombination. The TcRab7 gene was inserted into pTcGFPN (for localization experiments) and pTcCFPN (for co-localization experiments). The PAR 2 gene was inserted into pTcGFPN (for localization experiments) and pTcGFPH (for co-localization), while Tcpr29A and TcrL27 were inserted into pTcTAPN. The putative centrin was inserted into pTcMYCN (for localization experiments),

and into pTc6HN. For construction of GFPneo-CTRL and TAPneo-CTRL, first, a hypothetical T. cruzi gene (Tc00.1047053510877.30) was inserted in these vectors. Then, this genetic element was removed by restriction endonuclease digestion (SmaI), preserving the attB selleck chemicals recombination sites. Transfection of the parasites Epimastigote forms of T. cruzi Dm28c were grown at 28°C in liver infusion tryptose (LIT) medium, supplemented with 10% fetal calf serum (FCS), to a density of approximately 3 × 107 cells ml-1. Parasites were then harvested by centrifugation at 4,000 × g for 5 min at room temperature, washed once in phosphate-buffered-saline (PBS) and resuspended in 0.4 ml of electroporation

buffer pH 7.5 (140 mM NaCl, 25 mM HEPES, 0.74 mM Na2HPO4) to a density of 1 × 108 cells ml-1. Cells were then transferred to a 0.2 cm gap cuvette and 15 to Alvocidib solubility dmso 100 μg of DNA was added. For co-localization assays, 15 μg of each plasmid was used in the same cuvette. The mixture was placed on ice for 10 min and then subjected to 2 pulses of 450 V and 500 μF using the Gene Pulser II (Bio-Rad, Hercules, USA). After electroporation, cells were maintained on ice until being transferred into 4-10

ml of LIT medium containing 10% FCS, where they were incubated at 28°C. After 24 h of incubation, the antibiotic (hygromycin or G418) was added to an initial concentration of 125 μg ml-1. Then, 72 to 96 h after electroporation, cultures were diluted 1:10 and antibiotic concentrations were doubled. Stable resistant cells were obtained approximately 18 days after transfection. Southern blot analysis DNA extraction was performed according Angiogenesis inhibitor to Medina-Acosta & Cross [49], with some modifications. Briefly, 1 × 108 cells were pelleted, washed once with PBS and lysed with 1.5 ml of TELT buffer (50 mM Tris-HCl, pH 8.0, 62.5 mM EDTA, pH 8.0, 2.5 M LiCl and 4% Triton X-100). DNA was purified three times using phenol/chloroform/isoamilic alcohol (v/v). After that, DNA was precipitated by adding 100% ethanol (1:1, v/v), then washed three times with 1 ml of 70% ethanol, dried at 25°C and resuspended in 100 μl of TE containing 10 μg ml-1 RNase A. T. cruzi DNA (10 μg) was restriction digested with HindIII (Amersham Biosciences, Piscataway, USA) and was resolved on a 0.8% agarose gel in TBE buffer.