Fig. 7. Gel shift assay for APSI1 binding with the SRE sequence. a) Alignment of deduced amino acid sequences of APSI1, NtAPSI1-1, and NtAPSI1-2. The gaps are introduced to optimize the alignment. Residues found at the same positions as those in APSI1 are shown as white letters on black. The asterisks show the consensus amino acids among them. The sequences of RRP4 N-terminal region and OB-fold domain are indicated by lines. Gel shift assay detected the complex of SRE and the APSI1 (b) and NtAPSI1-1 (c) proteins using the LightShift Chemiluminescent RNA EMSA Kit. The biotin-labeled RNAs (bio-RNA, 20 fmol) were synthesized, incubated with the purified recombinant APSI1 and NtAPSI1-1 proteins (50 fmol), and electrophoresed directly on a 4% native polyacrylamide gel in 0.5× Tris/borate/EDTA. Unlabeled SRE RNAs (0, 200, 400, or 1,000 fmol) of were added as a competitor. The RNAs in the gels were visualized using LAS3000 Mini imaging system. The sequences of the synthesized RNA oligonucleotides (WT SRE and mutated SRE), positions of the RNA–protein complexes, and free RNA probes are indicated. The experiments were repeated at least 3 times and the representative results are shown.