EMSA results showing the effect of SNVs on NR2F1-binding activity from transfected nuclear extract (293T-NR2F1 ne) in the presence of increasing molar excess (25 to 50 to 100 to 200 as denoted by black slope) of pWT or pMut competitor ‘cold’ probes compared with conjugated ‘hot’ probe (pWT-IRDye 700). For each SNV: NR2F1 binding (upper gel); free probe (bottom gel, lower and upper bands reflect unannealed and annealed probe, respectively). In all five experiments, pWT shows decreasing NR2F1 binding and increasing free probes. Cluster A variant competitor probes (p3, p4 and p5) compete less well than pWT for NR2F1 binding (more NR2F1 shifted and less free probe available). Cluster B variants (p7–8 and p9), where no NR2F1 binding is expected, show no substantial effect. The same trend was observed in replicate experiments: WT = 11; p3 = 5; p4 = 8; p5 = 4; p7-8 = 3; and p9 = 7. Full gels are given in Source data
