Firstly, we performed a stepwise digitonin extraction of intact epimastigote cells. The pattern of Tc38 extraction was compared with those of cytosolic (PK), mitochondrial (CS), and glycosomal (HK) markers (Figure 3A). The Tc38 extraction curve clearly follows that of CS. It begins to be extracted at a digitonin concentration of 2.0 mg/mL, and at 5 mg/mL 39% of the protein still remained in the pellet. This pattern supports the hypothesis of a predominant mitochondrial localization of Tc38 in the cell.

Figure 3 Subcellular localization of Tc38 using biochemical approaches in T. cruzi I-BET-762 ic50 epimastigotes. (A) Digitonin extraction. Epimastigotes (125 mg per tube) were incubated with different digitonin concentrations (indicated on the abscissa) as described in Materials and Methods. Marker enzymes activities: hexokinase

(HK), citrate synthase (CS), and pyruvate kinase (PK). The amounts of Tc38 were determined by western analysis. (B) Subcellular fractionation. The experiment was carried out Afatinib ic50 using 3.3 g (wet weight) of parasites. Fractions are plotted in the order of their isolation, from left to right: nuclear (N), large granule (G), small granule (SG), microsomal (M) and supernatant (S). The ordinate represents relative specific activity (percentage of total activity/percentage of total protein). The abscissa indicates the cumulative protein content. The percentage of recovery for the marker enzymes: citrate synthase 70.9%, hexokinase 74.1%, tuclazepam cytochrome C reductase 43.6%, pyruvate kinase 85.3%, Tc38 61.1%. Error bars indicate the variation in band intensity seen by quantification of the western blot. Secondly, we

carried out subcellular fractionation experiments. They also showed that Tc38 is a mitochondrial protein since the highest specific activity was observed in the large granular fraction (Figure 3B). The recovery of CS activity in the nuclear fraction suggests a contamination of this fraction with mitochondrial proteins. Tc38 presents a complex pattern of distribution within the mitochondrion In order to address the subcellular localization of Tc38 with another approach we performed immunohistochemistry. The analysis of asynchronous epimastigote cultures showed a non-homogeneous Tc38 pattern (Figure 4). Parasites exhibit a widespread dotted distribution in an area that resembles the branched shape of the mitochondrion. In addition, 75.8 ± 0.5% (n = 500) cells present a strong Tc38 staining on the kinetoplast. As commonly seen in epimastigotes, DAPI brightly stains the “”disk”" shaped kinetoplast DNA and produces a weak signal in the rounded nuclear DNA. Although control experiments using nuclear protein antibodies verified the penetration of the antibodies into the nucleus (data not shown), we were unable to detect any consistent nuclear fluorescence from Tc38 in these preparations. Figure 4 Subcellular localization of Tc38 using immunohistochemical approaches in asynchronous cultures of T.