Polysomal Profiling of Cell Lysates

Polysomal profiling was performed according to previously described protocols (Bernabò et al., 2017 (link)). Briefly, the cells were treated with cycloheximide and then lysed in 300 μL of cold lysis buffer. The lysate was centrifuged at 4°C for 5min at 20.000 g to pellet cell debris. The cytoplasmic lysates loaded on a linear 15%–50% [w/v] sucrose gradient and centrifuged in a SW41Ti rotor (Beckman) for 1 h 40 min at 180.000 g at 4°C in a Beckman Optima Optima XPN-100 Ultracentrifuge. Fractions of 1 mL of volume, were then collected monitoring the absorbance at 254 nm with the UA-6 UV/VIS detector (Teledyne Isco).