Sucrose density gradient centrifugation

Sucrose density-gradient centrifugation was performed to analyze the cellular distribution of polysomes, 80S ribosomes and free 40S and 60S subunits. Cells were cultured in 100 mm dishes and transfected with siRNA and control for 24h, 48h and 72h. 15 minutes prior to lysis, cells were incubated with 100 μg/ ml cycloheximide (Sigma-Aldrich, Missouri, USA) to stall ribosomes on the mRNA strand. Lysis was performed on ice by washing cells in ice-cold PBS containing 100 μg/ ml cycloheximide followed by suspension in lysis buffer (20 mM HEPES pH 7.4, 15 mM MgCl₂, 200mM KCl, 1% Triton X-100, 2mM DTT and 100 μg/ml cycloheximide), and nuclei were removed by centrifugation (14000g, 10 min, 4°C). The supernatant was layered onto 15%-40% sucrose gradients (50 mM NH4Cl, 50 mM Tris-acetate pH 7.0, 12 mM MgCl2, 100μg/ ml cycloheximide and freshly added 1mM DTT) and centrifuged in a SW41Ti rotor (Beckman, Villepinte, France) for 150 min at 160000 g, 4°C without breaking. Sucrose density gradient profiles were analysed via an ISCO density gradient analyser unit, which analyses and simultaneously blots ribosomal distribution measured by an UA-6 detector with 254 nm filter (Teledyne ISCO, Nebraska, USA).

All fractions were precipitated with trichloroacetic acid overnight at -20°C to concentrate proteins for gel electrophoresis.