FIGURE 5.

TcMCU endogenous C-terminal tagging. A, PCR analysis using gDNA isolated from WT and TcMCU-3×HA cell lines. A DNA fragment was amplified in 3×HA-tagged epimastigotes (indicated with arrow), whereas the band is absent in WT. B, Western blotting analysis of WT and TcMCU-3×HA cell lines. Anti-HA antibodies detect TcMCU-3×HA (predicted size, 35 kDa), whereas the band is absent in WT parasites. Anti-α-tubulin antibody was used as a loading control. Antibodies are indicated on the right side of the blots, and molecular weights are on the left side. C, fluorescence microscopy of TcMCU-3×HA epimastigotes indicates localization of the endogenous tagged protein to the mitochondria. TcMCU-3×HA was detected with monoclonal anti-HA antibodies (green). Polyclonal antibodies anti-TbVDAC were used to label mitochondria (red). The merge shows co-localization in yellow. D, PCR analysis of TcFCaBP-3×c-Myc epimastigotes. A DNA fragment was amplified in c-Myc-tagged epimastigotes (indicated with arrow), whereas the band is absent in WT cells. E, Western blotting analysis of WT and TcMCU-3×c-Myc cell lines. Anti-c-Myc antibodies detect TcMCU-3×c-Myc (predicted size, 37 kDa), whereas the band is absent in WT epimastigotes. F, fluorescence microscopy of TcMCU-3×c-Myc epimastigotes indicates localization of the endogenous tagged protein to the mitochondria. TcMCU-3×c-Myc was detected with monoclonal anti-c-Myc antibody (green) or with polyclonal anti-TbVDAC antibodies (red). The merge shows co-localization in yellow. DIC images are shown in the left panel. Nucleus and kinetoplast were labeled with DAPI (blue). Bars, 10 μm.