Fig 1: Design and validation of bivalent ligands. A, bivalent ligands were formed via heterospecific coiled coil interactions of N- or C-terminal modified EGF (EN, EC) or NRG (NN, NC) fusion proteins. Thus, formation of bivalent ligands with two EGF domains, two NRG domains, or one EGF and one NRG domain is possible. B, possible mechanisms for the observed differences in monovalent NRG compared with bivalent NN, in which NN fosters homodimerization or homo-oligomerization of HER3 at the expense of heterodimers with HER2, are illustrated in the schematic. C, Western analysis is shown of pHER2 and pHER3 in lysates from NSCLC line H3255 cells stimulated for 15 min by monovalent NRG (NC) or bivalent NN (data are representative of two independent experiments). D, the schematic illustrates a model for EE binding that facilitates EGFR homodimerization or homo-oligomerization, leading to at least partial sequestration of EGFR from heterodimerization with HER2. E, HeLa cells were stimulated for 15 min with escalating doses of EGF or EE, lysed, and analyzed by Luminex technology for pan-pY-EGFR or pan-pY-HER2 (n = 3). AU, absorbance units. hTMSC were stimulated for 2 min with no ligand (F), 100 nm monovalent EGF (G), or 100 nm bivalent EE (H); cells were fixed, and EGFR receptors were identified by immunofluorescent staining (red). F, G, and H, nuclei were stained with DAPI (blue); scale bar = 30 µm, images are representative of 5 cells per condition (n = 5) from a representative experiment of 3 total.