Nebuffer for pk

4 μl of purified xCPEB4, full-length or N-terminal domain, at 2 mg/ml and 6 M urea were diluted with a urea-free buffer to a final volume of 12 μl in the presence of 3 μg of radiolabelled B1 or B1-123 RNA probe and 20 μg of tRNA. Samples were incubated 15 min at RT. After, proteins were diluted to a final volume of 100 μl in NEBuffer for PK (New England Biolabs) containing 200 μM ATP, incubated 30 min at 4°C and, when specified, phosphorylated with 500 U ERK2 and 1 μg Cdk1/cyclin B for 30 min at 30°C. Following phosphorylation, samples were loaded in Corning Costar Spin-X cellulose acetate 0.22 μm tube filters and centrifuged at 12,000 g for 1 min. Flow through was recovered and the filter column was washed with NEBuffer for PK. 4% of input and 20% of flow through were resolved in SDS-PAGE. Filters were exposed and developed with phosphorimager.

Purified xCPEB4 N-terminal domain, in 25 mM Tris-HCl pH 8, 100 mM NaCl, 5% glycerol, 5 mM MgCl₂ and 2 M urea buffer, was diluted in a non-containing urea buffer to a final protein concentration of 0.5 mg/ml and 0.2 M urea (other buffer components were kept at the same concentration). Samples were incubated at 4°C for 30 min before DLS analysis. For phosphorylation assays, proteins were diluted in NEBuffer for PK (New England Biolabs) containing 200 μM ATP, incubated on ice for 30 min and immediately phosphorylated with Cdk1/cyclin B (20 ng / μg xCPEB4 N-terminal domain) and ERK2 (10 U / μg xCPEB4 N-terminal domain) at 30°C for 30 min. Kinase reactions were stopped with 20 mM EDTA and kept at 4°C. 100 μl of sample were analysed by DLS with a Zetasizer Nano-S instrument (Malvern, Malvern, UK) at 25°C. Three measurements were obtained for each condition. Three independent experiments were performed.