Journal: Journal of Medicinal Chemistry
Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery
doi: 10.1021/acs.jmedchem.5c00782
Figure Lengend Snippet: Analysis of the affinity of multivalent ligands for HER2. (A) The expression level of HER2 in SKBR-3 and MCF-7 cell lines was analyzed by Western blotting. Tubulin levels were used as loading control. (B) The specificity of the interaction between GFPp_Affibody HER2:342 oligomers and HER2 receptor was confirmed by confocal microscopy. SKBR-3 and MCF-7 cells were incubated with 300 nM BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 for 30 min on ice. Nuclei were stained with NucBlue Live dye, and cells were fixed in a 4% paraformaldehyde solution. (C) GFPp_Affibody HER2:342 oligomers were incubated with the recombinant extracellular domain of HER2 (HER2.ecd-Fc) for 15 min at room temperature, and the formation of the ligand–receptor complex was confirmed by Native PAGE and Western blotting using anti-HER2 antibodies. Oligomers without added receptors were used as a control. (D) BLI analyses of the interaction between GFPp_Affibody HER2:342 oligomers and the HER2 receptor. HER2-Fc was immobilized on Protein A sensors, and the association and dissociation phases were monitored at different protein concentrations. A reference sensor without HER2-Fc was used as a control. (E) Microscopic analysis of the enhanced binding of multivalent variants to HER2. SKBR-3 cells were preincubated with DyLight 550-labeled monomeric Affibody HER2:342 for 10 min on ice, then equimolar concentrations of BiF HER2 , TriF HER2 , TetraF HER2 , or PentaF HER2 were added, and incubation was continued for 30 min. Cells incubated with monomeric proteins were used as a control. Nuclei were stained with NucBlue Live dye, and the cells were fixed in a 4% paraformaldehyde solution. Representative images from three independent experiments are shown. The scale bar is 20 μm.
Article Snippet: The NucBlue Reagent (Hoechst 33342) (# R37605 ), DyLight 550 NHS Ester (#62263) and HCS CellMask Stain Deep Red (#32721) were from Thermo Fisher Scientific.
Techniques: Expressing, Western Blot, Control, Confocal Microscopy, Incubation, Staining, Recombinant, Clear Native PAGE, Binding Assay, Labeling
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