SUnSET Assay for Protein Synthesis Measurement

SUnSET was performed using a previously described protocol (Hertz et al., 2014b (link); Shahi et al., 2016a (link)). Briefly, trophozoites (2 × 10⁶/ml) were incubated with 10 μg/ml puromycin (Sigma-Aldrich, St. louis, Mo, USA), a structural analog of tyrosyltRNA, for 20 min at 37°C. The trophozoites were lysed using 1% Igepal (Sigma) in PBS. Whole proteins were resolved on a 10% SDS-PAGE in SDS-PAGE running buffer. Proteins were electrotransferred in protein transfer buffer to nitrocellulose membranes. Loading equivalency was determined by immunoblotting using a 1:10,000 monoclonal α-tubulin antibody (DM1A clone, Sigma-Aldrich, St. louis, Mo, USA). puromycin was detected by immunoblotting using a 1:5,000 monoclonal puromycin antibody (12D10 clone, Millipore). After incubation with the primary antibody, the blots were incubated with 1:5,000 secondary antibody for 1 h at RT (Jackson ImmunoResearch, Enco Diagnostics, Israel), and then developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, USA). Protein quantification/synthesis was estimated from the intensity of the immunoreactive blots (densitometry) using Fiji software (Schindelin et al., 2012 (link)).