Fig. 2. TbRAB11 is a developmentally regulated GTPase. (A) Northern blot analysis demonstrating that BSF parasites express TbRAB11 mRNA at a higher level than PCF. Total RNA was isolated from 10⁷ cells and resolved on a formaldehyde gel. After transfer to a nylon membrane the blot was probed with the full length TbRAB11 ORF. Positions of RNA standards in kb are shown to the left and ethidium bromide stain of the rRNA bands shown as a control for loading equivalence at the base of the figure. (B) Western blot analysis with affinity purified anti-TbRAB11 generated in rabbits using full length recombinant protein as antigen. Fresh parasite cultures were resuspended in protein sample buffer, 10⁷ parasite equivalents loaded in each lane and resolved on a 12% SDS-polyacrylamide gel. After electrophoretic transfer to nitrocellulose, equivalence of loading was checked by Ponceau S staining. Molecular weight standards are indicated to the left in kDa. (C) GTPase assay using recombinant GST-TbRAB2 (lanes 1,3) and GST-TbRAB11 (lanes 2,4). Recombinant protein loaded with [-³²P]GTP was incubated at 37°C for 0 minutes (lanes 1,2) or for 60 minutes (lanes 3,4). After incubation [-³²P]GDP was separated from [-³²P]GTP by thin layer chromatography. (D) Graphical representation of GTPase activity of recombinant GST-TbRAB2 () and GST-TbRAB11 (). Recombinant protein loaded with [-³²P]GTP was incubated at 37°C for 0, 5, 10, 20, 40 or 60 minutes. After incubation [-³²P]GDP was separated from [-³²P]GTP by thin layer chromatography and the percent hydrolysis at each time point determined using phosphorimager analysis.