Fig. 4. Moderate overexpression of TbMBAP1. (A) The TbMBAP1 gene was expressed under the control of a constitutive (K) or tetracycline-inducible (Ti) procyclin promoter. Quantification of TbMBAP1 on immunoblots shows a moderate overexpression of three to fourfold compared to the wild type (wt). The red colour indicates the TbMBAP1 protein detected with anti-TbMBAP1-specific rabbit antiserum and IRDye 800 second reagent. As a loading control, a mouse anti-HSP60 antibody was detected with Alexa Fluor^TM 680-conjugated second antibody. Fluorescence quantification was done with the LiCor Odyssey system (see Materials and Methods). (B) Detection of overexpressed TbMBAP1 in the flagellar pocket. Cells were surface-labelled with AMCA-sulfo-NHS (blue). Detection of TbMBAP1 (green) and image processing were as in Fig. 3A. The lysosomal membrane protein p67 was visualized with a specific monoclonal antibody and Alexa-594-conjugated second antibody. While in wild-type cells TbMBAP fluorescence is absent from the flagellar pocket (see Fig. 3A), the protein can be detected in flagellar pocket (FP) membrane upon fourfold overexpression of the protein. The overall localization of TbMBAP1 between flagellar pocket and nucleus (N) is not affected.