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. 2016 Jul 12;67(17):5009–5027. doi: 10.1093/jxb/erw266

Fig. 11.

Fig. 11.

Gel shift assays to test interaction of WRKY18, WRKY40 or WRKY60 with the promoter of CRK5 gene. (A) WRKY18 binds to the ProCRK5-1, ProCRK5-2 and ProCRK5-3 fragments. 6×His-W18 indicates the purified 6×His-WRKY18 fusion protein; 6×His, the 6×His tag peptide served as negative control; Biotin-Probe, the biotin labeled CRK5 promoter fragments ProCRK5-1, ProCRK5-2, ProCRK5-3; mW1 and mW2/W3, the two mutant forms in W-boxes (W1 single mutation and W2/W3 double mutation) of biotin labeled ProCRK5-1 with W-box mutation at W1: TTGACT→TTAAAT, and W-box mutation at W2/W3: TTGACCTTGACT→TTAAACTTAAAT; Cold-Probe, the three unlabeled CRK5 promoter fragments; 50× and 200×, 50-fold or 200-fold cold-probe relative to the labeled probes added, respectively, for binding competition; Free-Probes, the labeled probes that do not bind the WRKY protein; WRKY18/ProCRK5, the shift bands of the complex of WRKY18 protein with corresponding ProCRK5 fragments. Each experiment was repeated three times with the same results. (B) WRKY40 binds to the ProCRK5-1, ProCRK5-2 fragments, but does not bind to the ProCRK5-3 sequence. 6×His-W40 denotes the purified 6×His-WRKY40 fusion protein; WRKY40/ProCRK5, the shift bands of the complex of WRKY40 protein with the corresponding ProCRK5 fragments. Other symbols are the same as described above in (A). Each experiment was repeated three times with the same results. (C) WRKY60 does not bind to any of the three promoter segments of CRK5 gene. 6×His-W60 denotes the purified 6×His-WRKY60 fusion protein, and other symbols are the same as described above in (A). Each experiment was repeated three times with the same results.