Fig. 2

EMSA for the specific binding of BmILF to the i-motif structure. The i-motif probe was synthesized and refolded into an i-motif structure at pH 4.0, 6.0 and 8.0. The ssDNA is the unfolded sequence. The cold probe is the un-labeled i-motif probe. The sequence of the mutated probe is shown in Table 1. The linear free probe is the same DNA fragment that did not form an advanced structure during the annealing cooling process. EMSA for the binding of recombinant BmILF to the i-motif probe of BmPOUM2a and BGIBMGA003213b at pH 4.00 in the 1 × binding solution (20 μl, 2.5% glycerol, 0.05% NP-40, 5 mM MgCl₂, 4 mM EDTA, recombinant BmILF protein and biotinylated end-labeled probe) at room temperature for 20 min. The running buffer contained 0.04 M Tris, 0.04 M H₃BO₃, 0.001 M EDTA-2Na and was filtered with 0.22-µm pore-size filter. EMSA for the binding of recombinant BmILF to the i-motif structure of BmPOUM2 (c) and BGIBMGA003213 (d) or the linear ssDNA probe at pH 4.0, 6.0 and 8.0. EMSA for the binding of recombinant BmILF to different DNA motifs on BmPOUM2 (e) and BGIBMGA003213 (f). The positions of the labeled i-motif-containing probe, labeled ssDNA probe, labeled bound i-motif and BmILF are shown by the arrows. g CD analysis of the complex of BmILF and i-motif at pH 4.0. The sequences of all the probes used in this figure are listed in Table 1