cruzi cells during a single transfection experiment using pTcGW vectors (Figure 4). There was also no correlation between

fluorescence intensity (Figure 4) and cytometry analysis data (Figure 3C). This absence of correlation was possibly caused by find more differences in exposure times and contrast (Figure 4). Indeed, we obtained the subcellular localization of a putative centrin of T. cruzi using the vector AZD6244 concentration pTcMYCN (Additional file 3 – Figure S2). This protein is related to centrosome and was located in epimastigotes near to kinetoplast in agreement with personal communication (Preti, H.). Figure 4 Subcellular localization of Tc Rab7 and PAR 2 in T. cruzi using p Tc GW vectors. Fluorescence microscopy of epimastigotes transfected with GFPneo-CTRL, GFPneo-PAR2, GFPneo-Rab7, GFPhyg-PAR2 and

CFPneo-Rab7. The merged frame was composed by “”GFP”" and “”DAPI”" images overlap. The DAPI frame in the last row was replaced by a frame containing the cyan fluorescence-Rab7 construct (*), in which a red signal was used. The “”#”" frame contains a merger of DAPI/GFPhyg-PAR2/CFPneo-Rab7. Fluorescent proteins have been employed for subcellular localization in several types of organisms. This approach has some advantages: it is rapid and avoids the use of antibodies. However, in some cases, this technique may result in protein misallocation, due to at least two factors: (i) overexpression of recombinant proteins [37]; and (ii) interference of N- or C-terminal fusions with the localization signals [38, 39]. Sirolimus price To circumvent these Cytoskeletal Signaling inhibitor problems, the platform described here was conceived for use with various strategies. First, recombinant vectors can be used without the pol I promoter, which may diminish expression of recombinant proteins. Moreover, the IRs might be promoting different gene

expression levels with the constructs in this study; thus, each IR could then be replaced by a non regulated or regulated IR, enabling standardized levels of expression or life cycle-specific expression, respectively. Our group is currently employing deep sequence and proteomic analysis to select specific intergenic regions for use in pTcGW vectors. Also, the analysis of gene sequences to detect particular localization signals may help to choose between N- or C-terminal fusions. The constructs in this study were designed for N-terminal fusions, but they can be modified quickly to generate C-terminal tags. Tandem affinity purification The tandem affinity purification (TAP) tag [40] comprises two repeated B domain of protein A (able to bind IgG), plus the site for TEV protease and the calmodulin binding peptide (CBP). The main reason for using a tandem purification approach is to avoid false positives. Two genes already described in the literature, Tcpr29A [41] and TcrL27 [42] were inserted into pTcTAPN.