Fig. 1.

Linker length has limited impact on bivalent affibody-mediated pHER3 inhibition and HER3 downregulation. a Schematic representing multivalent affibody constructs with 3 or 64 amino acid (aa) linker size. b Purified protein products are displayed on a Coomassie stained gel loaded at 10 μg/lane. Bivalent affibodies with 3 aa linker and 64 aa linker ligands have respective theoretical molecular weights of 14.4 and 19.1 kDa. c OvCAR8 cells were treated with the indicated concentrations of bivalent affibody 3 or 64 aa linker or 100 nM pan-HER kinase inhibitor (total inhibitor), for 30 min, then stimulated with 10 nM NRG for 10 min, lysed, and probed for pHER3, pAkt, and β-actin by immunoblot. d OvCAR8 cells were treated with 10 nM bivalent affibody with 3 and 64 aa linker, lysed at the indicated time points, and probed by immunoblot for HER3 and β-actin. All multivalent ligand concentrations refer to the concentration of individual affibody domain. Results shown are representative of three independent experiments. All blots were cropped; blots for pHER3 and pAkt are presented at different exposures for clarity