Polysome profile analysis

Cells were washed with cold HBSS, scrape-harvested directly into lysis buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl₂, 1 mM DTT, 100 µg/ml cycloheximide, 100 µg/ml heparin, and 1% NP-40 made in DEPC-treated water), and supplemented with RNAsin Plus inhibitor (Promega) and HALT phosphatase and protease inhibitors (Thermo Fisher Scientific). Lysates were rotated at 4°C for 15 min, cleared by centrifugation for 10 min at 12,000 g, and supernatants were loaded on preformed 17.5–50% sucrose gradients made in gradient buffer (10 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl₂, and 1 mM DTT). Samples were centrifuged in a Beckman SW140 Ti rotor for 2.5 h at 35,000 rpm and then eluted using a Brandel bottom-piercing apparatus connected to an ISCO UV monitor, which measured the eluate at OD 254.