Fig. 3.

HER3 downregulation by bivalent affibodies is a specific phenomenon. a Schematic of various affibody constructs: monovalent HER3 (7.6 kDa), HER3 bivalent affibody with 3 or 64 amino acid linker (14.4 and 19.1 kDa), monovalent EGFR (7.6 kDa), HER3 EGFR bivalent bispecific affibody with 3 and 64 aa linker (14.3 and 18.9 kDa), EGFR bivalent affibody with 3 or 64 aa linker (14.4 and 18.2 kDa), bivalent bispecific HER3 wild-type affibody with 64 aa linker (19.1 kDa), monovalent HER3 affibody with 64 aa linker tail (12.5 kDa). b Purified protein products are displayed on a Coomassie stained gel loaded at 10 μg/lane with respective molecular weights listed in Figure 3a. c OvCAR8 cells were treated with 10 nM of the indicated affibody construct or media alone (no treatment), lysed at the indicated time points and probed by immunoblot for HER3 and β-actin or d EGFR and β-actin. All multivalent ligand concentrations refer to the concentration of individual affibody domains. Results shown are representative of three independent experiments. All blots were cropped