Figure 3

TbRab23 is a flagellar protein. (A) Subcellular fractionation of trypanosomes corroborates the location of tagged TbRab23. 1x10⁷ BSF cells were incubated in PMN buffer to separate flagellar, basal body and cytoskeletal-associated proteins from the rest of the proteome ('Soluble'). 10⁷ cell equivalents were collected at each stage for SDS-PAGE; 'Flagellar cytoskeleton' denotes pellet containing isolated axoneme, flagellum attachment zone (FAZ), paraflagellar rod (PFR), four specialized microtubules and basal body proteins. The majority of TbRab23HA was recovered in the first fraction, but a small amount was also present in the same fraction as TbPFR indicating the presence of TbRab23HA on the flagellar cytoskeleton. The cytoskeleton and flagellar cytoskeleton were enriched in β-tubulin as expected. For presentation purposes, empty wells have been removed from the figure and are represented by gaps. The isolation has been performed twice with similar results. (B) Purified flagella were processed for IF using anti-HA counterstained with Alexa-568 revealing the presence of TbRab23 at a structure associated with the flagellum on basal bodies. Scale bar 2 μm. IF on flagella extracted from cells expressing a late endosomal protein TbVps26HA, did not produce a signal (data not shown).