Irdye 700 fluorophore

GATA6, mutant GATA6, or empty pcDNA3.1(+)-HA vector were expressed using the TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI) according to the manufacturer’s protocol. Oligo DNA corresponding to wild-type or mutant CR1 and CR2 (the same sequences as oligo DNA pulldown assay) were synthesized with and without the IRDye® 700 fluorophore (Integrated DNA Technologies, Coralville, IA) and annealed to generate labeled probes and unlabeled competitor DNA. In vitro synthesized protein (2 μL) was incubated with 1 μg of poly dI-dC in binding buffer (10 mM Tris pH 8.0, 50 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 5 mM MgCl2, 1 mg/ml BSA) at room temperature for 10 minutes. For antibody treatment, pre-binding was performed in the presence of active or heat-inactivated anti-human GATA6 antibody (AF1700, R&D Systems Inc.). IRDye® 700-labelled probe (100 fmol) was then added and the binding reaction proceeded at room temperature for 15 minutes. DNA-protein complexes were resolved on a 6% non-denaturing polyacrylamide gel in 0.5x TBE (40 mM Tris pH 8.3, 45 mM boric acid, and 1 mM EDTA) at room temperature. Fluorescence was detected using an Odyssey CLx imager (LI-COR Biosciences, Lincoln, NE).

NKX2-5 HD binding was evaluated using 40 bp sequences centered on the SNP and an additional 20 bp sequence for IRDye^® 700 fluorophore conjugation(Integrated DNA Technologies). All sequences were ordered in IDT and are available in Supplementary Table 2. Reference and alternate oligonucleotides were labeled with the IR-700 fluorophore through a primer extension reaction. Binding reactions were performed in binding buffer (50 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 10% glycerol) and 5 nM fluorescently labeled dsDNA. Binding reactions were incubated for 30 min at 30°C, then 30 min at room temperature before. The 6% polyacrylamide gel in 0.5x TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.4) was pre-runned at 85 V for 15 min, loaded at 30 V, and resolved at 75 V for 1.5 h at 4°C. Gels were imaged with Azure^® Sapphire Bio-molecular Imager at 658 nm/710 nm excitation and emission.