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Additional file 1: Fig. S1. Internalization of various FGF1-SA oligomers via FGFR1-mediated endocytosis.A. Live cell imaging of endocytosis of monomeric FGF1E-AviTag-Biot and FGF1E-AviTag-Biot in combination with different SA variants. U2OS-R1 cells were incubated on ice for 40 min with Alexa Fluor 488 C5 maleimide-labeled FGF1E-AviTag-Biot alone or in the presence of SA variants of different valency (from 1 to 3). Then, cells were transferred to 37°C and imaged live for 60 min using a spinning disk confocal microscope. Images taken at the indicated time points are shown. The scale bar represents 50 m. B. Quantitative analysis of endocytosis of FGF1E-AviTag-Biot alone or in combination with different variants of streptavidin. Mean values from three live cell imaging experiments +/-SEM are shown. Fig. S2. Development of the tetrameric MMAE-FGF2v-Biot-SA-4AA - B. FGF2V was conjugated to the cytotoxic compound MMAE via N-terminal cysteine flanked by two lysines. Then, the conjugated protein was biotinylated using sortase A and assembled with tetrameric SA-4A to yield a cytotoxic tetrameric conjugate. The efficiency of conjugation, biotinylation and correctness of complex assembly were confirmed by SDS-PAGE with CBB staining (A) and western blotting with antibodies directed against FGF2 (B). Thermal denaturation of the SDS-PAGE samples was skipped to preserve the tetrameric form of proteins. C. Biotinylation of the cytotoxic conjugate was confirmed by BLI by measuring the interaction of MMAE-FGF2V and MMAE-FGF2V-Biot with streptavidin-bearing SAX2 biosensors. Association and dissociation profiles were measured. D. The cytotoxic potential of the tetrameric conjugate MMAE-FGF2v-Biot-SA-4A was evaluated in U2OS-R1 cell line. Cells were treated with MMAE-FGF2V-Biot in the presence or absence of SA-4A at various concentrations for 96 h. Then, cells viability was assessed with the Presto Blue assay. Results are mean values from three experiments +/-SEM. Fig. S3. Development of the tetrameric MMAE-AffibodyHER2-Biot-SA-4A targeting HER2 receptor.A. AffibodyHER2 was conjugated with cytotoxic MMAE via an N-terminal KCK motif. Then, conjugated protein was biotinylated with using sortase A and assembled with SA-4A to obtain a tetrameric MMAE-AffibodyHER2-Biot-SA-4A conjugate. The purity and identity of the proteins at each reaction step were verified by SDS-PAGE with CBB staining. To preserve the tetrameric form of the protein during SDS-PAGE, the thermal denaturation step was omitted. B. BLI comparison of MMAE-AffibodyHER2 and MMAE-AffibodyHER2-Biot binding to streptavidin using SAX2 biosensors. Association and dissociation profiles were measured. C. The cytotoxic potential of monomeric MMAE-AffibodyHER2 and tetrameric MMAE-AffibodyHER2-Biot-SA-4A was measured in the SKBR3 cell line. Cells were treated with MMAE-AffibodyHER2 or MMAE-AffibodyHER2-Biot-SA-4A at various concentrations for 96 h. Then, cell viability was assessed with the Presto Blue assay. Results are mean values from three experiments +/-SEM. 4x – monomeric MMAE-AffibodyHER2 was used at four times higher concentrations in the experiments to provide cells with equal molar concentrations of drug and targeting molecule.