Biochemical characterisation of the fractions obtained during the <i>T</i>. <i>foetus</i> 20S proteasome isolation procedure.

<p>(A) Coomassie brilliant blue-stained 12.5% SDS–PAGE of TE, F1, F2, F3 and F4-fractions (1 X 10⁹ cells). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129165#pone.0129165.s005" target="_blank">S5 Fig</a> for a description of each fraction. S, molecular weight standard. Similar results were obtained for PS and EFF. (B) Immunoblot and densitometric analyses using an anti-proteasome antibody. The antibody reacted specifically with two bands near 27-kDa in each fraction. The results of densitometry were normalised to the intensity of bands in the TE fraction and are expressed as the means of relative densitometric units ± SD across three independent experiments. The proteasomal protein level increased approximately 4.5-fold in the F3-fraction. **p<0.01; ***p<0.001 compared to TE-fraction. Similar results were obtained for PS and EFF. (C) Fluorogenic substrate assay of proteasome activity of each fraction obtained from PS (light grey bars) and EFF (dark grey bars). The caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (CT-L) activities were measured by spectrofluorometry using the fluorogenic substrates Z-LLE-AMC, Z-ARR-AMC and Z-LLL-AMC, respectively. Data are expressed as means of fluorescence units ± SD across three independent experiments performed in triplicate. The peptidase activities of proteasome from EFF were significantly higher than those from PS parasites. **p<0.01; ***p<0.001 compared to PS. In both parasite forms, the higher proteasome activity levels were found in the F3-fraction. (D) Effects of 20 μM lactacystin (Lac), 100 μM E-64 and 100 μM TPCK on the T-L, C-L and CT-L activities of F3-fractions obtained from PS (light grey bars) and EFF (dark grey bars). Data are expressed as means of relative percentage of control (without inhibitors) ± SD across three independent experiments performed in triplicate. In both forms, the peptidase activities were inhibited by lactacystin only. The proteasomal activities of the EFF were significantly more susceptible to lactacystin when compared to those of the PS. Similar results were obtained for other fractions. **p<0.01; ***p<0.001 compared to PS.