Polysomal Profiling for Translational Regulation

Polysomal profiling was done as described (Tcherkezian et al., 2014 (link); Culjkovic-Kraljacic et al., 2016 (link)). Briefly, cells were treated with cyclohexamide (100 µg/ml, Sigma Aldrich, Cat# C7698) 10 min before harvesting and lysates were prepared using polysome lysis buffer (15 mM Tris pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1% Triton X-100, 100 g/ml cyclohexamide, 1 mM DTT, 400 U/ml RNase inhibitors and protease inhibitors (Sigma Aldrich, Cat# 11697498001). Equal amounts (10 mg) of protein lysates were layered on a 20–50% linear sucrose gradient (20% and 50% sucrose solutions in 15 mM Tris pH 7.4, 15 mM MgCl2, 150 mM NaCl, 1 mM DTT, 100 µg/Ml cyclohexamide and 20 U/ml RNase inhibitors), mixed on Gradient Station IP Biocomp and centrifuged in a Beckman SW41Ti rotor at 92,000 g for 3 hr at 4˚C. Following centrifugation, polysomal fractions were collected by continuously monitoring and recording the A254 on a Gradient Station IP (Biocomp) attached to a UV-MII (GE Healthcare, CA) spectrophotometer. RNAs were isolated from polysomal fractions using TRIzol reagent. RNAs from each fraction were monitored using RT-qPCR.