FIGURE 10.

Analysis of TcP5CDH over distinct parasite life stages. A, expression levels of TcP5CDH in the predominant life stages of T. cruzi. Noninfected CHO-K₁ cells served as controls. Total cell-free extracts from each parasitic stage were used as enzymatic sources in the TcP5CDH activity assay (left axis). RNA preparations from the stages noted above were used in quantitative real time PCR assays (quantitative RT-PCR). Comparisons were performed using the 2^−ΔΔCt method and normalized relative to TcGAPDH expression levels. Values plotted correspond to the levels of fold change compared with the indicated epimastigote stage (right axis). The differences among the samples were calculated using one-way analysis of variance and Tukey's post test considering a p value < 0.05 as significant. B, Western blot analysis for TcP5CDH levels in the above-listed parasitic life stages. Protein sample preparations are described under “Experimental Procedures,” and equivalent amounts (40 μg) of each preparation were loaded per lane. The samples were subjected to SDS-PAGE (10%), transferred to nitrocellulose membranes, and probed with polyclonal antibodies raised against TcP5CDH (1:2000) and TcGADPH (1:2500). The latter did not react with GAPDH isoforms from the CHO-K₁ cells used in our assay conditions.