Figure 3
I-17 causes eIF2α phosphorylation and translational initiation impairment. (A) Total extracts of exponentially growing T. cruzi epimastigotes (NT), submitted to nutritional stress (TAU), or treated with 10 µM of I-17 for 4 hours were analyzed by immunoblotting using antibodies against the phosphorylated threonine 169 of eIF2α (T169[P]), anti-TceIF2α, and anti-HSP70. The original figures are depicted in as Supplementary Figure 2. (B) Ratio between phosphorylated (T169[P]) and the total TceIF2α intensity signal. The values are mean ± standard deviation of triplicate experiments and **indicates p < 0.01 calculated using the Student’s t-test. (C) Total extracts of exponentially growing T. brucei procyclics (NT) were submitted to 10 µM of I-17 for 4 or 24 hours and analyzed by immunoblotting using antibodies against the phosphorylated threonine 169 of eIF2α (T169[P]), anti-TceIF2α used to detect TbeIF2α, and anti-HSP70. (D) Polysome profiles in sucrose gradients measured at 254 nm of epimastigotes non-treated (NT) or treated with I-17 at 10 µM. The migrating position of the ribosomal subunits (40S and 60S), monosomes (80S) and polysomes (P) is indicated in each panel. The P/M ratios were obtained by measuring the graphic area under both fractions.
