Ribosome-Polysome Profile Analysis

Ribosome–polysome profile analysis was performed as described previously (16 (link)) with modifications. Namely, twenty A260 units of extract were layered onto 7–47% sucrose (w/w) gradient and centrifuged at 4°C in SW28 rotor (Beckman Coulter, Brea, CA, USA) at ω²t = 1.8 × 10¹¹. For 60S and 40S ratio quantification ribosome-polysome profile analysis was carried out with following modifications. The addition of cycloheximide to the cell cultures was omitted and Mg²⁺ was excluded from all buffers and sucrose gradients. Twenty A₂₆₀ units of extract were layered onto 10–25% sucrose (w/w) gradient and centrifuged at 4°C in SW28 rotor (Beckman Coulter) at ω²t = 3.4 × 10¹¹. Areas under monosome (80S), polysome, 60S and 40S peaks were quantified by ImageJ and corresponding ratios were calculated. The average and standard deviations for at least three biological replicates were calculated. Statistical significance was determined by the unpaired two sample Student's t test.