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. Author manuscript; available in PMC: 2015 Aug 25.
Published in final edited form as: Mol Cell Endocrinol. 2014 Jul 6;394(0):47–58. doi: 10.1016/j.mce.2014.06.021

Figure 1.

Figure 1

Met and FISC are sufficient for in vitro binding of JHRE. A) Schematic diagram of E. coli-expressed N-terminal Met fused with GST-tag and FISC with His-tag. B) Coomassie staining of the purified recombinant Met and FISC. The asterisks (*) indicate protein bands with expected sizes. C) Gel-shift assay with the recombinant proteins. Met and FISC, or either protein alone, were incubated with the DNA probe (AaET JHRE1, 5′-CCATCCCACACGCGAAGACGATAAAACCA-3′) in the presence or absence of 10−6 M JH-III for 20 minutes followed by electrophoresis. For competition, 50-fold molar excess of unlabeled specific (S) or non-specific (N) competitor DNA, was mixed with proteins for 20 minutes before addition of the probe. D) Super-shift assay. Antibodies for the GST-tag (GST) or His-tag (His6) were included in the DNA-binding reactions. Non-specific rabbit IgG was used as a control. The experiments were repeated three times with similar results. Representative autoradiographs are shown.