Fig. 2.

Effect of RNAi against candidate genes on proliferation of BSF T. brucei. (A) Cumulative proliferation curves for each of four candidates. RNAi cell lines were cultured in the presence (+Tet) or absence (−Tet) of 1 μg/ml tetracycline. The experiment for each candidate was carried out in triplicate with essentially identical results, and included the SMB cell line as a negative control. (B) Validation of targeting specificity. The mRNA levels of three genes, Tb11.01.2640 (ERAP32), Tb927.7.3870 and Tb927.2.5140 was measured by qRT-PCR and normalized to tubulin. Results are presented as the mean ± SD from two independent experiments. Down-regulation of Tb11.01.8120 (ERAP18) was addressed by Western blotting against an HA-tagged version of the gene product expressed from an integrated pXS5 plasmid. Lysates were probed with anti-HA and also anti-BiP as loading control. (C) Numbers of nuclei (N) and kinetoplasts (K) in ERAP32 and ERAP18 RNAi cell lines. DNA was visualized by DAPI staining followed by examination under a fluorescence microscope. Error bars represent mean ± SD from analysis of 300 cells in three independent experiments.