Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model

E. histolytica trophozoites (3x105) harvested in exponential growth phase were transferred to 25 cm² cell culture flasks and grown at 37°C for 24 h. Then, parasites were exposed to irradiation in a 254 nm UV-transilluminator (BioDoc-It Imaging System (UVP)) for 30 min at 4°C (Valdés et al., 2014) and cytoplasmic (CE) and nuclear (NE) extracts were obtained using the NE-PER Kit (Thermo Scientific™) according to manufacturer instructions. Immunoprecipitation (IP) assays were performed using Dynabeads protein A/G (Invitrogen) following manufacturer recommendations. Briefly, Dynabead magnetic beads (25 µl) were coupled with 10 µg of anti-HsCFIm25 polyclonal antibody (GeneTex, GTX115535) and anti-EhCFIm25 polyclonal antibody, previously produced and validated by our group (Pezet-Valdez et al., 2013) for 30 min at room temperature. Then, beads-antibody complex was incubated with NE (100 µg) for 2 h at 37°C followed by three washes with wash buffer (20 mM Tris, pH 7.5; 0.5M NaCl and 0.05% Tween 20). Finally, magnetic bead- antibodies-proteins complex was resuspended in elution buffer (0.1 M glycine, pH 2.0) for 2 minutes at room temperature to dissociate the complex; the tube was placed on the magnet and the supernatant containing eluted antibody and bound proteins was collected.