Molecular and Electrophysiological Characterization of a Novel Cation Channel of Trypanosoma cruzi
Figure 3
Functional yeast complementation with TcCat.
A–D. TcCat expression, as analyzed by immunofluorescence, at different times after induction. Yeast were collected at the indicated times and incubated with anti-TcCat antibodies (green) and anti-vacuolar H+ ATPase (red) as a control for proper permeabilization. Nuclei were DAPI stained (blue). Left panels are DIC images, right panels are anti-TcCat stained cells and central panels are merge immunofluorescence images. Bars = 10 µm. E. Western blot analysis of yeast homogenate with specific anti-TcCat antibody. Lanes, 1: control non-complemented mutant yeast, 2: wild-type strain complemented with TcCat, 3: mutant strain complemented with TcCat, 4: TcCat recombinant protein. F. Growth-assay of complemented yeast in SC ura- galactose agar plates. Serial dilutions of initial cultures at OD600 = 0.6 were incubated for 72 h at 30°C. WT corresponds to wild type strain, MpYES2 is the mutant transformed with the empty vector pYES2, MC represents the mutant strain transformed with TcCat-pYES2 construct.