FIGURE 3.
Characterization of LdRab1 and its mutants. A, GTP binding of purified LdRab1:WT and its mutants was detected using an [α-32P]GTP overlay assay. LdRab5:WT and GST proteins were used as control. B, GTPase activity of LdRab1 and its mutants was determined as described under “Experimental Procedures.” C, to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania, cell lysates were analyzed by Western blotting using anti-GFP antibody. Untransfected Leishmania was used as control. D, to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania, cell lysates were analyzed by Western blotting using anti-LdRab1 antibody. Untransfected Leishmania was used as control. E, the same membrane was exposed for a longer duration to detect endogenous Rab1. F, to determine the localization of Rab1:WT and its mutants in Leishmania, cells were transfected with indicated constructs to overexpress the respective protein in Leishmania as GFP fusion protein. Cells were visualized in a LSM 510 Meta confocal microscope. Green, localization of the indicated LdRab1; blue, nucleus (Nu). Results are representative of three independent preparations.