Gradient profiler v1

Wild-type and mutant embryos were dechorionated with pronase (1 mg/mL). 200 dechorionated embryos per genotype were lysed in 550 μL of lysis buffer (20 mM Tris-Cl pH 7.5, 30 mM MgCl₂, 100 mM NaCl, 0.25% Igepal-630 (v/v), 100 μg/mL cycloheximide, 0.5 mM dithiothreitol (DTT), and 1 mg/mL heparin). Embryo lysates were incubated for 10 min on ice and centrifuged for 10 min at 4°C with 21,000 xg. 200 μL of clarified lysates were loaded onto a continuous 10-50% (w/v) sucrose gradient prepared in TMS buffer (20 mM Tris-Cl pH 7.5, 5 mM MgCl₂, 140 mM NaCl). Gradients were centrifuged in a SW40 Ti rotor (Beckman) at 4 °C and 35,000 rpm for 165 min. Polysome gradients were analyzed using a gradient station (BioComp) coupled to a Model Triax™ Flow Cell detector (FC-2). The precise location of the fractions along the UV-tracing was monitored using the Gradient profiler v1.25 (BioComp) software. Polysome gradients were normalized prior to quantification for baseline differences. The area under the curve was calculated for the defined fractions. Polysome-to-monosome ratio was calculated by dividing the value of the polysome fraction by the obtained value of the 80S fraction.