a Structure of the tetravalent peptides synthesized on a cellulose membrane is shown. The density of tetravalent peptides on the membrane and the spacer length was optimized for the screening using Stx as follows. The density was set to 100% by using Fmoc-β-Ala-OH without Boc-β-Ala-OH for the first peptide synthesis cycle, and the number of aminohexanoic acid residues (U; spacer length) was set to one (upper panel). Met or Ala at position three or five of MMA-tet, respectively, was replaced by the indicated amino acid (βA; beta-Ala). The tetravalent peptides synthesized on a membrane were blotted with 125I-Stx1a (left panel). The screening was performed three times (Supplementary Fig. 1). Eight Stx1a-binding motifs (shaded in the right panel) were identified. b Structure of the tetravalent peptide and the identified motifs are shown. c MMβA-tet most efficiently inhibited the cytotoxicity of Stx1a and Stx2a. Vero cells were treated with Stx for 72 h in the presence of each peptide. Data are presented as a percentage of the control value (mean ± standard error (SE), n = 3). d AlphaScreen assay to examine the binding between MMβA-tet and the B-subunit or its mutant. Data are presented as signal intensity (mean ± SE, n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 (compared with wild type by Dunnett’s test).