Figure 3. Intracellular distribution of TcMVK.

(A) TcMVK partially co-localized with aldolase. Immunofluorescence images acquired with confocal microscope show that TcMVK is localized in glycosomes of T. cruzi epimastigotes (EP), extracellular amastigotes (EA) and metacyclic trypomastigotes (MT). Differential interference contrast (DIC); DAPI (blue); rabbit anti-aldolase (green); mouse anti-TcMVK (red) and colocalized pixels (CP, white). Bar: 4 μm. Images are representative of three independent experiments. (B) Western blot analysis of EA (left panel) and MT (right panel) conditioned medium confirming secretion of TcMVK; EAs and MTs were incubated for 16 h in RPMI at 37 °C (1 × 10⁸ parasites/ml). After centrifugation, the supernatant from 1 × 10⁸ parasites was precipitated with trichloroacetic acid and subjected to SDS-PAGE (13% gel). The cell pellet was solubilized in Laemmli buffer and the equivalent of 1 × 10⁸ parasites was submitted to SDS-PAGE (13%). Anti-MVK was raised in mice using recombinant TcMVK and used at a dilution of 1:1000. EA and MT: whole cellular lysate (centrifugation pellet) from extracellular amastigotes and metacyclic trypomastigotes respectively; 0 h: Incubation time point zero; 16 h: Extracellular amastigotes or metacyclic trypomastigotes 16 h conditioned medium; (−): Empty lanes; 34 kDa: TcMVK expected molecular mass. These results are representative of two independent experiments.