Polysomal profile Clausole campione

Polysomal profile. Growing cells were lysed using a glass douncer in 50 mM Tris HCl pH 7.8, 240 mM KCl, 10 mM MgSO4, 5mM DTT, 250 mM sucrose, 2% Triton X-100, 90 μg/ml cicloheximide, 30U/ml RNasin. After centrifugation at 39000 r.p.m. for 3 hours at 4 °C, the equivalent of two- hundred micrograms of RNA was loaded on a 15-55% sucrose gradient dissolved in 25 mM Tris HCl pH 7.4, 25 mM NaCl, 5 mM MgCl2, 1 mM DTT and spun at 260.000 g for 3h30min with SW41Ti swing rotor (Xxxxxxx Xxxxxxx). The gradient was then analyzed by continuous flow absorbance at 254 nm, recorded by BioLogic LP software (BioRad). Peaks for 40S, 60S, 80S and polysomes were quantified. For dissociation studies, total extracts of REN cells were incubated 2 minutes at 37 °C, with 5µg of recombinant eIF6 protein or matched controls (PBS; denatured protein), and separated on a 7-45% sucrose gradient. Extracts containing up to 200 micrograms of RNA were loaded on a 7–45% (w/v) sucrose gradient containing 50 mM Tris-acetate pH 7.5, 50 mM NH4Cl, 12 mM MgCl2 and 1 mM DTT, and centrifuged in a Xxxxxxx XX00 Ti rotor for 3h30min at 260.000 g. The gradient was analyzed as above. In addition, individual fractions were collected. Fractions were precipitated with 10% trichloroacetic acid (TCA), separated on SDS-PAGE and analyzed by Western blot. For microRNAs profiling on polysomal profile of REN cell, they were lysed using a glass douncer in 50 mM Tris HCl pH 7.8, 240 mM KCl, 10 mM MgSO4, 5mM DTT, 250 mM sucrose, 2% Triton X-100, 90 μg/ml cicloheximide, 30U/ml RNasin ± 30mM EDTA. Following clearing, RNA was loaded on a 10-50% sucrose gradient dissolved in 25 mM Tris HCl pH 7.4, 25 mM NaCl, 5 mM MgCl2, 1 mM DTT ± 30mM EDTA, and spun at 260.000 g for 3h30min with SW41Ti swing rotor (Xxxxxxx Xxxxxxx). The gradient was then analyzed by continuous flow absorbance at 254 nm, recorded by BioLogic LP software (BioRad). Peaks for 40S, 60S, 80S and polysomes were quantified. Fractions of 1 ml were collected.