cruzi genome. Figure 1 Southern blot analysis of transfected T. cruzi cells. Lanes Luminespib solubility dmso represent HindIII-digested: genomic DNA from

T. cruzi wild type (WT), from T. cruzi transfected with the TAPneo-Tcpr29A plasmid (29A) and TAPneo-Tcpr29A isolated plasmid (Control). The neomycin resistance marker (NEO) and the tandem affinity purification tag (TAP) were used as probes. 1 Kb Plus DNA Ladder (Invitrogen) was used as the molecular weight marker. This result was not surprising, as plasmid integration into the ribosomal locus has previously been shown in other constructs in which a ribosomal promoter was used [3, 34]. Besides, there is also the possibility that the vectors 10058-F4 solubility dmso were integrated into other areas of the T. cruzi genome, such as the ubiquitin locus, as the IRs (TcUIR) for this locus were present in three copies in our constructs. Analysis of mRNA levels To analyze mRNA levels for the GFP-fused recombinant protein in T. cruzi transfected with

GFPneo-CTRL, GFPneo-Rab7 or GFPneo-PAR2, we performed real-time RT-PCR using oligonucleotides to amplify GFP. GFPneo-CTRL mRNA levels were approximately nine-fold higher than those of GFPneo-Rab7 and were six-fold higher than those of GFPneo-PAR2 find more (Figure 2). To better understand cell resistance without fluorescence, we quantified NEO mRNA levels in the same populations for which GFP mRNA levels were analyzed. Levels of NEO mRNA were greater than GFP mRNA in GFPneo-Rab7-transfected T. cruzi (Figure 2). Differences occurred despite all vectors containing a similar structure (i.e., IR sequences, resistance marker, protein tag and promoter). Also, although GFP-fused mRNAs are distinct, this is not the case for NEO mRNAs. This is an interesting point that still needs to be addressed. Figure 2 Levels of GFP-fused and NEO recombinant mRNAs in T. cruzi. IKBKE The Y-axis indicates the level of GFP

and NEO mRNA quantified by real-time RT-PCR using populations of cells transfected with GFPneo-Rab7, GFPneo-PAR2 and GFPneo-CTRL. Detection of recombinant proteins and FACS analysis of transfected T. cruzi To confirm the presence of recombinant proteins in transfected T. cruzi, western blot assays were performed using antibodies against the tags. The bands in Figure 3B correspond to the expected molecular weight of the PAR 2 and TcRab7 with addition of the GFP tag and the sequence for the attB1 site. Detection of TcrL27 and Tcpr29A recombinant proteins (using anti-calmodulin binding peptide antibody) is shown in the “”Tandem affinity purification”" section, while the centrin recombinant protein used with c-myc and polyhistidine tags (using anti-c-myc and anti-histidine antibodies) are shown in Additional file 1 – Figure S1. Predicted molecular weight of native proteins TcrL27, Tcpr29A, PAR 2, centrin and TcRab7, including the protein tags are described in Additional file 2 – Table S1.