To select a peptidomimetic ligand against ITGA5, overlapping sequences (12 amino acids long with 8–amino acid overlaps) from human FN-III domains 9 and 10 were designed and displayed on a cellular membrane. Domains 9 and 10 of FN were chosen to design peptidomimetics, as these domains were reported to be responsible for binding to the α5β1 receptor, as shown by the docking experiments (26). The interaction studies were performed against human recombinant integrin α5β1 receptor (R&D Systems), the bound proteins were transferred to another membrane, and ITGA5 was detected with antibodies. AV3 (Arg-Tyr-Tyr-Arg-Ile-Thr-Tyr) and AV3-PEG6-5FAM (AV3-FAM) were custom-synthesized by China Peptide Co. Ltd. (Shanghai, China). The % purities were 98% for AV3 and 95% for AV3-PEG6-5FAM, as assessed by reversed-phase high-performance liquid chromatography (HPLC, analytical). The products were stored at −20°C.

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