Recombinant ybx1

Final concentration 2 nM of radiolabelled RNA was adjusted to 5 ml with Milli-Q water, incubated 1 min at 95 °C and cooled on ice for 2 min. RNA was allowed to fold for 30 min at 37 °C in binding buffer (mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.1 mM CaCl₂, 1 mM DTT, 0.1 mg/ml BSA, 0.4 mg/ml fragmented yeast RNA (Sigma), 5% glycerol, 0.025% bromophenol blue and 0.025% xylene cyanol). Recombinant YBX1 (Abcam) was added at the concentrations indicated and the binding reaction was carried out at 30 °C for 30 min. Samples were loaded on a non-denaturing 0.7% agarose gel in cold 1× TBE buffer. After 2 h at 120 V of gel electrophoresis, the gels were vacuum dried for 90 min at 80 °C and exposed to an Amersham Hyperfilm ECL film.

One microgram of recombinant YBX1 (Abcam) was incubated with 20 ng of recombinant RSK1 protein (R&D) and the corresponding in vitro-transcribed RNA in kinase buffer (25 mM TRIS/HCl, pH 7.5, 5 mM glycerolphosphate, 2 mM DTT, 10 mM MgCl₂, 50 µM ATP, 10 µCi γ-32P (Perkin Elmer), protease inhibitor mixture Complete (Roche), phosSTOP (Roche)) for 15 min at 30 °C. Laemmli buffer was added to stop the reaction and boiled at 95 °C for 5 min. Proteins were separated by electrophoresis in 4–12% NuPAGE Bis–Tris gels (Invitrogen). Subsequently, the gel was dried at 80 °C for 1 h and then exposed to an Amersham Hyperfilm ECL film. The band intensities were measured using ImageJ/Fiji software v2.0.0.