Sucrose gradients were used to separate subpolysomal and polysomal ribosomes. Ten to 50% (w/v) sucrose gradients were prepared in gradient buffer [300 mM NaCl2, 15 mM MgCl2, 15 mM tris-HCl (pH 7.5), 1 mM DTT, and cycloheximide (0.1 mg/ml)]. Cells were seeded on a 15-cm dish, washed in phosphate-buffered saline (PBS)–cycloheximide (100 μg/ml), and scraped into lysis buffer [300 mM NaCl2, 15 mM MgCl2, 15 mM tris-HCl (pH 7.5), 1 mM DTT, 0.2 M sucrose, cycloheximide (0.1 mg/ml), 0.5% IGEPAL, and 5 μl of RNasin per 1 ml]. Lysates were incubated on ice for 3 min before pelleting cells at 1300g for 5 min. The supernatant was layered onto the gradient and centrifuged at 38,000 rpm (acceleration, 9; deceleration, 6) for 2 hours at 4°C using a Beckman Coulter ultracentrifuge. Gradients were fractionated using a gradient fractionation system (Presearch Ltd.), and fractions were collected at 1-min intervals using a Foxy Jr. collection system (Presearch Ltd.), at a flow rate of 1 ml/min. Absorbance was measured constantly at 254 nm using a UA-6 UV-visible detector (Presearch Ltd.). The relative rate of translation was estimated by calculating a ratio of polysomes/subpolysomes by measuring the area under the curve within the subpolysomes (fractions 1 to 5) and the polysomes (fractions 6 to 10).

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