Recombinant LI-cadherin Protein Production

All LI-cadherin constructs were expressed as previously described^{9 (link)}. Briefly, Expi293F cells (Thermo Fisher Scientific) were transfected with the pcDNA3.4 vector encoding LI-cadherin sequence and C-terminal Myc-tag, NSAVD sequence, and 6xHis-tag , following the manufacturer’s protocol. Cells were cultured at 37 °C and 8.0% CO₂ for three days after transfection. The supernatant was collected by centrifuging the cell culture for 15 min at 1500 rpm and was dialyzed against a solution of 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 3 mM CaCl₂. Proteins were purified by immobilized metal affinity chromatography using Ni–NTA Agarose (Qiagen), followed by size exclusion chromatography using the HiLoad 26/600 Superdex 200 pg column (Cytiva) at 4 °C equilibrated in buffer A (10 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, and 3 mM CaCl₂). Unless otherwise indicated, samples were dialyzed in buffer A before analysis, and the filtered dialysis buffer was used for assays.