Perfect match (wild type, WT) or mutated (MUT) oligonucleotides to the predicted NKX6.1 binding site within the 8-kb deletion were designed (Supplementary Table 8) and used in an EMSA assay. Non-radioactive chemiluminescent detection was employed, and specifically the Light Shift Chemiluminescent EMSA Kit (Fisher-Thermo Scientific, UK). Briefly, labelled or unlabeled oligonucleotides were incubated with cell lysates of HEK293 cells expressing the human NKX6.1 protein (Novus Biologicals, EU), in the presence or absence of anα-NKX6.1 antibody (Abcam, UK). The DNA-protein complexes were run on 4% DNA retardation polyacrylamide gels (Invitrogen, UK), transferred to a nylon membrane using iBlot DNA transfer stacks and iBlot transfer system (Invitrogen, UK), and the resulting chemiluminesce detected by exposure to an X-ray film (Kodak, UK).
