(A) RIG-I E373Q binding site at ES7L-A was used to design a 60b hairpin RNA (ES hairpin). RNA secondary structure was determined with the RNAfold webserver (). (B) Electrophoretic mobility shift assays of RIG-I or RIG-I E373Q incubated with 24mer dsRNA. Complexes were pre-formed at 37 °C for 20 min, separated on agarose gels and stained with GelRed. Free RNA bands were quantified using ImageJ. Protein concentrations (from left to right): 0, 0.1 µM, 0.3 µM, 0.5 µM, 0.7 µM, 1 µM, 1.5 µM and 2 µM. *: unbound RNA, **: protein:RNA complexes. (C) Electrophoretic mobility shift assays of RIG-I, RIG-I E373Q or RIG-I C268F incubated with ES hairpin RNA. Complexes were pre-formed, separated and stained as in panel B. Protein concentrations (from left to right): 0, 0.5 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM and 10 µM. *: unbound RNA, **: protein:RNA complexes.
DOI: http://dx.doi.org/10.7554/eLife.10859.012