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. 2015 Oct 23;290(50):29993–30005. doi: 10.1074/jbc.M115.670018

FIGURE 2.

FIGURE 2.

Purification and characterization of LdRab1. A, purification of GST-LdRab1. GST-LdRab1 was purified as described under “Experimental Procedures” and analyzed by SDS-PAGE. Lane 1, protein marker; lane 2, uninduced lysate of E. coli; lane 3, induced lysate of E. coli; lane 4, purified GST-LdRab1. B, specificity of LdRab1 antibody. To prepare anti-LdRab1 antibody, GST tag was removed by thrombin cleavage, and mice were immunized with LdRab1 as described under “Experimental Procedures.” The specificity of anti-LdRab1 antibody was checked by Western blotting using purified GST-LdRab1, GST-LdRab5, and GST-LdRab7. Results are representative of three independent preparations. C, to determine the endogenous localization of LdRab1 in Leishmania, cells were fixed and permeabilized with saponin as described under “Experimental Procedures.” Permeabilized cells were probed with LdRab1, washed, and visualized using Alexa Fluor 594-labeled goat anti-mouse secondary antibody. Red, localization of LdRab1; blue, nucleus (Nu). Cells were viewed in an LSM 510 Meta confocal microscope. Results are representative of three independent preparations.