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. 2022 Dec 2;13:7448. doi: 10.1038/s41467-022-34991-2

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022

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Fig. 2 — A Sequence of wildtype and mutant CE probes for EMSA. B EMSA showing migration of recombinant NKX2-3 and COUP-TFII bound to the CE probe (left) and to CE_ΔC_A probe that lacks the COUP-TFII “A” site (right). Co-incubation of NKX2-3 and COUP-TFII yields a distinct band (blue arrows) reflecting an NKX2-3:COUP-TFII DNA complex that is supershifted by anti-NKX2-3 antibody but not by control antibody. C EMSA of protein complexes formed by incubation of the indicated TFs with a mutant CE probe lacking the NKX2-3 and the COUP-TFII “A” binding sites (CE_ΔN_ΔC_A). The probe binds COUP-TFII (lane 7) and not NKX2-3 (lane 2), but seeds a COUP-TFII:NKX2-3:DNA complex (blue arrow) when co-incubated with both TFs. The heterodimer-DNA complex is supershifted by anti-NKX2-3 antibody (red star). NKX2-3^ΔTN failed to heterodimerize with COUP-TFII (lane 6). D Disruption of the NKX2-3:COUP-TFII heterodimer-DNA complex by a competing NKX2-3 TN domain peptide (WT) or by a scrambled peptide (SP). Data are expressed as percent of probe bound by NKX2-3:COUP-TFII heterodimer. E Madcam1 expression in TNFα-treated DN-MAML bEnd.3 cells stably overexpressing Nkx2-3 or the Nkx2-3 TN domain, as evaluated by real-time PCR. Values are mean ± SEM of three biological replicates. Two tailed t-test, paired. **: p value < 0.01. F Proximity ligation assay of NKX2-3 and COUP-TFII in TNFα-treated DN-MAML bEnd.3 cells stained for MAdCAM1 (blue) and ERG (green), with the proximity ligation signal shown in red. Inset shows a negative control with isotype-matched primary antibodies. Scale bar: 20 $μ$ m.