Fig. 5. Inducible, strong overexpression of TbMBAP1. A cell line expressing TbMBAP1 under the control of the tetracycline-inducible T7 promoter was grown in the absence of tetracycline for 4 days, which reduced the enzyme to the wild-type level (normalized to 1 at time 0 hours in A and B). The culture was then induced with tetracycline for 24 hours, subsequently deprived of the inducer for 24 hours and finally grown for 12 days in the presence of tetracycline. (A) Immunoblot showing the increase in TbMBAP1 within 24 hours (left) and 12 days (right) of induction, which was quantified relative to the uninduced cells as indicated by the numbers above the blot. Asterisk: cells were grown for 12 days in the absence of tetracycline. TbHSP60 was used as a loading control. (B) Relative TbMBAP1 activity at various time points during the same experiment. Asterisk: cells grown for 24 hours without tetracycline. (C) 3D-Fluorescence microscopy of cells before (0 hours) and after 3, 5 and 20 hours of induction. 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). For each time point one representative cell is shown as an isosurface rendered, deconvolved 3D image. The white asterisks indicate the location of the flagellar pocket.