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. 2007 Sep 1;6(11):1964–1978. doi: 10.1128/EC.00279-07

FIG. 2.

FIG. 2.

(A) TbUBP1/2 protein expression in transgenic T. brucei. Total cell lysates from bloodstream-form or procyclic-form trypanosomes were subject to denaturing SDS-gel electrophoresis and blotted; detection was done with a polyclonal antiserum to TcUBP1. The blot was also stained with antibody to the cytosolic protein CSM, which migrates at about 100 kDa. The positions of molecular mass markers are on the left. The transgenes in the different cell lines are designed for tetracycline-inducible expression as follows: TbUBP2 overexpression (lanes 1 to 3 and lanes 7 to 9) and TbUBP1/2 RNAi (lanes 4 to 6 and lanes 10 to 12). The times after tetracycline addition are indicated: time zero indicates no tetracycline (lanes 1, 4, 7, and 10); 16-h (bloodstream) and 24-h (procyclic) time points with tetracycline are lanes 2, 5, 8, and11, and the patterns after 24 h (bloodstream) or 48 h (procyclic) with tetracycline are shown in lanes 3, 6, 9, and 12. The effects on TbUBP1/2 are also shown. “+” indicates normal expression from endogenous genes; upward arrowheads denote overexpression, and downward arrowheads denote depletion. Likely identities of the protein bands are indicated in the center between the images. (B) Staining pattern of a Western blot using extracts of different trypanosome strains and anti-TcUBP1 antibody. The 247 and Antat 1.1 strains have pleomorphic bloodstream forms, whereas the 927 strain is more culture adapted but can still differentiate readily. Two different bloodstream 427 isolates were checked; the one labeled 427a can differentiate into growing procyclic trypanosomes in culture, whereas the one labeled 427 cannot. (C) Bloodstream-form trypanosomes with inducible expression of myc-tagged proteins were incubated with tetracycline to induce expression. Lanes 1 and 4 are extracts from control cells without the transgene, lanes 2 and 5 are extracts from transgenic parasites grown without tetracycline, and lanes 3 and 6 are extracts from cells grown for 24 h in 100 ng of tetracycline/ml. Proteins were separated by SDS-PAGE and detected on Western blots with anti-TcUBP1 antiserum. Bands which reacted with the anti-myc antibody (not shown) are indicated with stars. Quantitation of the bands indicated that the myc-tagged TbUBP2 was ∼2-fold overexpressed in bloodstream forms. (D) Procyclic-form trypanosomes with inducible expression of myc-tagged proteins incubated with or without tetracycline. Details are as in panel C. Lane 1 is an extract from control cells without the transgene; lanes 2 and 4 are extracts from transgenic parasites grown without tetracycline, and lanes 3 and 5 are extracts from cells grown for 24 h in 100 ng of tetracycline/ml. Quantitation of the bands indicated that the myc-tagged TbUBP2 was ∼2-fold overexpressed, relative to cells without the transgene, and myc-TbUBP1 was 3-fold overexpressed. (E) Expression of N-terminally in situ-tagged TbUBP1 and TbUBP2. A sequence encoding a V5 tag was integrated into the genome in order to give an in-frame fusion with either TbUBP1 (lane 2) or TbUBP2 (lane 3); cells without the tag are in lane 1. Details are as for panel A except that the detecting antibody was anti-V5 for the left-hand panel. Detection with anti-TcUBP1 antibody followed the V5 detection; lanes 1, 2, and 3 are here marked 4, 5, and 6, respectively, for clarity of description in the text. Three more cell lines for each tag gave indistinguishable results. The stars mark the positions of the tagged proteins. (F) Quantitation of TbUBP1 and TbUBP2 protein levels in procyclic trypanosomes. Lanes 1 to 4 contained various numbers of trypanosomes, and lanes 5 to 8 contained serial dilutions of purified GST-tagged TbUBP2.