Fig. 6. TbCMF proteins localize along, and are stably associated with, the flagellum. (A) Anti-TPN western blot on whole-cell lysates (L), detergent-solubilized proteins (S1) and detergent-extracted cytoskeletons prepared (Hill et al., 2000) from a trypanosome cell line harboring a Tet-inducible TPN-GFP fusion protein. The TPN-GFP fusion protein is expressed at levels equivalent to endogenous TPN and is quantitatively associated with the cytoskeleton. (B-F) Fluorescence microscopy shows that TPN-GFP is correctly localized to the flagellum (arrows) in live cells (C-D) and in cytoskeletons (E-F). (B) Live cells exhibit background autofluorescence that does not overlap with the flagellum and is also evident in the 29-13 parent cell line. As a negative control, GFP alone was used. Although fluorescence was evident in the cell body of live cells, there was no fluorescence in the flagellum of detergent-extracted cytoskeletons (supplementary material Fig. S2). (G) Fluorescence microscopy of detergent-extracted cytoskeletons prepared from cell lines harboring the indicated Tet-inducible TbCMF-GFP fusion proteins. In all cases, the GFP fusions are localized along the flagellum (arrows). (H) Anti-GFP western blots of whole cell lysates (L), detergent-solubilized proteins (S1) and detergent-extracted cytoskeletons (P1) prepared from induced (+) and uninduced (-) TbCMF-GFP strains. The asterisk indicates a secondary band at 100 kDa that reacts with the anti-GFP primary antibody. Bars, 5 µm (B); 10 µm (G).