The molecular weights observed on SDS-PAGE were slightly higher this website than those expected based on the deduced amino acid sequences of TcKap4 and TcKap6. This difference may result from the basic nature of these proteins. Figure 3 Expression of recombinant TcKAPs in E. coli. The TcKAP4 (A) and PI3K inhibitor TcKap6 (B) were expressed in E. coli M15 strain following induction with 1 mM IPTG for 3 h. Immunoblotting assays of non-induced (1) and induced (2) bacterial extract using anti-polyhistidine antibody confirmed the expression of recombinant TcKAPs. Figure 4 Detection of TcKAPs in T. cruzi. Western blot analyses of (1) epimastigote, (2) amastigote/intermediate form and (3)

trypomastigote extracts of T. cruzi, using anti-TcKAP4 (A) or anti-TcKAP6

(B) serum. Both antisera recognized a single polypeptide in all developmental stages of the parasite. The kinetoplast ultrastructure in T. cruzi and distribution of TcKAPs The TcKAP antisera were also employed to determine the subcellular location of TcKAPs in T. cruzi. It is worth mentioning that the kinetoplast of this parasite undergoes morphological changes during the protozoon life cycle; epimastigotes and amastigotes have tightly packed kDNA fibers condensed within the kinetoplast disk, whereas trypomastigotes have a more relaxed organization of kDNA, which is enclosed in a rounded structure. During the transformation of amastigotes in trypomastigotes inside the mammalian cell, changes occur Mizoribine order in Decitabine solubility dmso the general organization of the protozoa, in special in the kinetoplast structure. The population of intracellular parasites does not differentiate in perfect synchrony, thus at a certain time of the differentiation process, some transitional stages

between amastigotes and trypomastigotes can be found in the same cell [20]. For this reason, the amastigotes used in our assays, which were released after disruption of LLC-MK2 cells, were mixed with intermediate forms. The kinetoplast of these intermediate forms is enlarged in relation to the disk observed in amastigotes, presenting the DNA fibers densely packed in the central area, but less condensed at the periphery. In order to analyze the distribution of TcKAPs in all developmental stages of T. cruzi, we carried out immunolabelling assays using TcKAP antisera in epimastigotes, amastigotes/intermediate forms and trypomastigotes. Both antisera specifically recognized the kinetoplast of all developmental stages of T. cruzi (figures 5 and 6). However, the distribution of these proteins within the kinetoplast depended on the developmental form of the parasite. In epimastigotas and amastigotes, TcKap4 and TcKap6 were distributed throughout the kDNA network (figures 5A–H for TcKAP4 and 6A–H for TcKAP6), consistent with findings for C.