Luciferase control mrna

Prior to lysis, cells were treated with 100 µg/mL cycloheximide (CHX) for 5 min and washed with cold PBS containing 100 µg/mL CHX. Cells were harvested in 1 ml of cold PBS containing 100 µg/mL CHX. Cell pellets were resuspended in hypotonic buffer (5 mM Tris [pH 7.5], 2.5 mM MgCl₂, 1.5 mM KCl, 0.3% CHAPS, 20 mM glycerophosphate, 10 mM NaF, 10 mM sodium pyrophosphate and EDTA-free protease inhibitor) containing 100 µg/mL CHX, 2 mM DTT, and 200 U/ml RNasin (Promega) by vortexing for 5 s. Supernatants were collected by centrifugation for 10 min at 14,000 rpm, 4°C, and the A₂₆₀ of lysates was measured to normalize RNA levels. Lysates of 30 O.D.₂₆₀ were loaded on 10–50% sucrose density gradients (100 mM HEPES [pH 7.6], 1M KCl, 50 mM MgCl₂, 100 µg/mL CHX, 200 U/ml RNasin, and EDTA-free protease inhibitor) and centrifuged at 35,000 rpm for 3 hr at 4°C. Gradients were fractionated and the optical density at 254 nm was recorded using an ISCO fractionator (Teledyne ISCO). For immunoblotting, the fractions were concentrated by Vivaspin concentrator (Sartorius). For quantitative PCR normalization, 5 ng of luciferase control mRNA (Promega) was added to each fraction. RNA was extracted using TRIzol and cDNA was synthesized by SuperScript III First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions.