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. 2022 Nov 19;23(22):14405. doi: 10.3390/ijms232214405

Figure 4.

Figure 4

Determination of binding capacity of recombinant nano-HER2-E3 associated with eGFP-K3 or 488-C-K3 on HER2 overexpressing cells. (A) Cartoon representation of the heterotetramer biomolecule composed of nano-HER2-E3/eGFP-K3 binding to HER2 cell surface receptor and the positive control nano-HER2-E3-C covalently labeled with Alexa-Fluor-488 maleimide. (B) Determination of relative binding affinities of nano-HER2-E3 alone or either co-associated with eGFP-K3 or 488-C-K3 on breast cancer cells. HCC1954 cells were incubated with increasing concentrations of nano-HER2-E3-C-488, nano-HER2-E3/eGFP-K3 or nano-HER2-E3/488-C-K3, or with eGFP-K3 alone as a control. Following incubation, fluorescence was measured by flow cytometry. The relative mean fluorescence was plotted against nanobody concentration (nM), and the apparent KD value was determined using sigmoidal fitting with R software (DRC package, R Core Team GNU GPLu2 version 4.2.1 Columbia, SC, USA).