Fig. 2: DLK1 binds the TGF-β superfamily receptor ACVR2B.

A Analysis of the Bioplex 3.0 interactome¹⁵ identified 42 potential DLK1 binding partners. The pie chart shows the fractions of candidate proteins containing extracellular domains (magenta) and intracellular proteins (teal). ACVR2B (bold) is the only known cell surface receptor among extracellular domain-containing proteins. B Confocal microscopy images depicting wild type U2OS cells or DLK1-expressing U2OS cells stained with recombinant ACVR2B-Fc protein. ACVR2B-Fc binding was detected with an anti-Fc Alexa Fluor 488 antibody, the contours of the cells were visualized by actin staining (magenta) using phalloidin 647, and nuclei were counterstained using DAPI (blue). The images are represented as maximum projections of 5 z-slices taken 0.8 µm apart. Scale, 20 µm. The experiment was independently repeated three times. C SPR was used to determine the steady-state binding affinity between DLK1(N-EGF6) and ACVR2B-Fc. The DLK1 protein was injected over a sensor chip containing immobilized ACVR2B-Fc and the data was fitted to a 1:1 binding model. RU = resonance units. The associated SPR sensograms for this data are shown in Supplementary Fig. 4A. D Confocal microscopy images depicting the staining of U2OS cells overexpressing ACVR2B coupled to a GFP^SPARK (green) tag, with DLK1-Fc protein (magenta). DLK1 binding was detected using an anti-Fc Alexa Fluor 647 antibody. Nuclei counterstained with DAPI (blue). Scale, 20 µm. The experiment was independently repeated two times. E Flow cytometry histograms depicting the binding of DLK1-Fc protein to yeast expressing ACVR2B and eleven other TGF-β superfamily receptors. The experiment was independently repeated two times. Source data are provided as a Source Data file.