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. 2020 Aug 27;12(9):2433. doi: 10.3390/cancers12092433

Figure 4.

Figure 4

NR4A3 and MYB cooperate and interact. (A) Western blot analysis. Sample identifiers are shown at top, antibodies used to probe the blot are indicated at right. An actin loading control is shown at the bottom. The fusions that were detected (‘+’) or not detected (‘–’) by either RNA-seq or FISH are indicated along the bottom (blanks were not tested). High expression of NR4A3 protein was detected in all samples with t(4;9), but not in the normal samples (right) or the sample with t(2;4), which expressed the NR4A2 gene instead. Two distinct NR4A3 proteins were detected; the main form (approximately 68kD, lower band) and an isoform (approximately 80kD, upper band). MSANTD3 protein was detected in the two samples that also had fusions activating that gene (390A2 and 670H1); (B) NR4A3 and Myb cooperate. HeLa cells were transfected with the NuRE promoter–luciferase vector plus plasmids expressing NR4A3 alone or with different Myb constructs, as indicated. The Myb proteins are diagrammed at the bottom. Additional controls are in Figure S5; (C) NR4A3 pull-down assays. The NR4A3 protein is diagrammed at top, including the DNA binding (red) and ligand binding (blue) domains. GST fusion proteins used for pull-down assays are diagrammed. Left panel: Cos-7 cells were co-transfected with plasmids expressing HA-tagged NR4A3 or Myb, protein complexes were immunoprecipitated using control IgG or anti-HA tag antibodies, and the complexes were detected by western blot using anti-Myb antibodies. Right panel: Myb protein was in vitro translated (IVT) and incubated with GST alone or GST-NR4A3 fusion proteins as indicated. The GST-bound complexes were isolated and assayed by western blot using anti-Myb antibodies; (D) Myb pull-down assays. The Myb protein is diagrammed, with several conserved domains shaded. GST fusion protein constructs are diagrammed. HA-tagged NR4A3 protein generated by in vitro translation was incubated with GST-Myb fusion proteins. The GST-bound complexes were isolated and assayed by western blot using anti-HA antibodies.