ELISA.

To evaluate integrin or ICAM1 binding to immobilized BR_Srr2 or BR_Srr1 regions, ELISA assays were performed as follows: proteins were coated at 5 μg/mL overnight. After extensive wash and saturation with BSA (Probumin, MilliporeSigma), 50 μL of ligand proteins (human integrins, ICAM1, or negative control BSA) were diluted at the specified concentrations in PBS containing 1% BSA (Probumin) and incubated 2 hours at 37°C. Integrin binding was assessed using antibodies listed in Supplemental Table 4. Binding was detected using o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich) or 3,3′,5,5′-tetramethylbenzidine (TMB) (Thermo Fisher Scientific). Values of the negative control obtained with BSA (Probumin) were subtracted from the signal. The effect of divalent cations on the interaction between BR_Srr2 and integrins was tested by ELISA with the same protocol as above in the presence of 1 mM Mn²⁺ or 1 mM Ca²⁺, as specified. Similarly, competition experiments were carried out by incubating 10 μg of integrins with immobilized BR_Srr2 in the presence of increasing concentrations of mimetic peptides (RGDS, RGDfV, or P11).