Fig. 6. Tetracycline-inducible knockdown by RNAi shows that TbMBAP1 is essential for growth of T. brucei bloodstream cells. (A) Growth in the presence (black circles), absence (white circles) of TbMBAP1-dsRNA. (B) Immunoblot showing the decrease in TbMBAP1 within 20 hours of RNAi. HSP60 was used as a loading control. (C) 3D-Fluorescence microscopy of cells before (0) and after 4, 7 and 12 hours of induction. Upper panel: The cell surface is visualized by AMCA-sulfo-NHS staining (grey), TbMBAP1 by anti-C-terminus antibodies and Alexa Fluor^TM 594-labeled secondary antibodies (red), and endosomes (green) by the autofluorescence of the marker protein EP:GFP (Engstler and Boshart, 2004). The yellow colour indicates colocalization of GFP and TbMBAP1. The arrows point to the flagellar pocket. In the lower panels only TbMBAP1 is shown. For each time point, images of representative cells were chosen. For the ease of visualization the deconvolved 3D data were processed by isosurface rendering.