Electrophoretic mobility shift assay.

The RNA fragment of Regnase-1 3′-UTR (nt194-212, IBA GmbH) was radioactively labeled using T4 polynucleotide kinase (Thermo Fisher Scientific) and [γ³²P] ATP (Hartmann Analytic) at 37 °C for 30 min. The reaction was stopped at 75 °C for 10 min. Sepharose spin columns (NucAway; Invitrogen) were used to separate RNA from free nucleotides. Radioactively labeled RNA (6 nM), proteins (GST–regnase-1^{aa1-452;D141N}, Roquin-1^aa2-440) and tRNA competitor (30 μg ml⁻¹) were incubated in HEPES/NaCl/MgCl₂ buffer (10 mM HEPES (pH 7.5), 150 mM NaCl and 2 mM MgCl₂) and 4% glycerol in a final volume of 20 μl for 30 min at 20 °C. Samples were resolved by native TBE–PAGE (4% polyacrylamide and 1× TBE buffer) or by gradient NativePAGE 4–16% Bis-Tris (Invitrogen) gels. Gels were analyzed using Fuji imaging plates exposed in the FLA-5100, after 10 min incubation in fixing solution (30% (v/v) methanol and 10% (v/v) acetic acid) and vacuum drying.