Ubiquitination in treated T. cruzi parasites

T. cruzi epimastigotes in the logarithmic growth phase (3×106 cells mL-1) were incubated for 12h with bortezomib (1.8 µM), GNF6702 (2.9 µM) or compound 1 (24 µM), equivalent to 8× the EC50 values of each compound. Controls were incubated in the presence of diluent (DMSO). Cells were harvested by centrifugation (1912g, 15 min, 4 °C) and washed with ice-cold PBS (1912g, 5 min, 4 °C), and finally, the cell pellets were resuspended in 1.5 mL of ice-cold lysis buffer (1 mM EDTA, 1 mM DTT, 100 μM TLCK, and 1× Roche EDTA-free cOmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer, pH 7.4). Cell suspensions were submitted to 3 freeze–thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and then lysed using the One ShotTM Cell disruptor (Constant Systems, UK) at 30 kpsi.