4.13. Polysome profiles

Cells were lysed in detergent lysis buffer A (10 mM Tris‐HCl at pH 7.4, 10 mM NaCl, 1.5 mM MgCl₂, 0.5% [v/v] Triton X‐100, 0.5% [w/v] deoxycholate, 1% [v/v] Tween‐20, 100 μg/ml cycloheximide) with complete EDTA‐free protease inhibitors (Roche) and 0.5 U/ml RNase inhibitor (Promega) and incubated for 10 min on ice. Lysates were cleared in a microfuge. Equal amounts (typically 10–20 A254 U) were applied to a 10%–50% (w/v) sucrose gradient in 11 ml of buffer B (10 mM Tris‐HCl at pH 7.4, 75 mM KCl, 1.5 mM MgCl₂) and centrifuged (Beckman SW41 rotor) at 207,570 g for 80 min at 4°C. Samples were unloaded using a Brandel gradient fractionator, the polysome profiles were detected using a UV monitor (Gilson) at A254, and fractions were collected. For analysis of polysome‐associated mRNAs, polysomal fractions were pooled and RNA was purified using the RNeasy Mini Kit (Qiagen). RNA was reverse‐transcribed with QScript enzyme (Quanta) according to the manufacturer's protocol, and the cDNA was used as a template for PCR. Primer oligos used were as follows:

p16: CGGTCGGAGGCCGATCCAG / GCGCCGTGGAGCAGCAGCAGCT

p21: CCTGTCACTGTCTTGTACCCT / GCGTTTGGAGTGGTAGAAATCT

IL1α: AGTGCTGCTGAAGGAGATGCCTGA / CCCCTGCCAAGCACACCCAGTA

β‐actin: CATGTACGTTGCTATCCAGGC / CTCCTTAATGTCACGCACGAT