Mutations to ICAM-4 affect adhesion of HT1080 cells. Adhesion of HT1080 cells to native and mutated ICAM-4Fc in a microplate adhesion assay. (A) Representative binding assays depicting various mutant ICAM-4Fc (♦) and native ICAM-4Fc (□) are shown, 2 from the A strand of domain 1 (F18A and V20T), 2 from the G strand of domain 1 (R92E and T95V), 2 other mutations resulting in a reduction of adhesion (W66A and K118E), 2 mutations leading to an increase in ICAM-4 adhesion (N160A and T162V), and 2 mutations that had no affect on adhesion (W77A and T162V). The proteins were used at 20, 10, 5, 1, or 0.25 μg/mL, and the results are shown as the percentage of input cells bound □ SD (n = 3 [mutants] or n = 2 [native protein]). (B) Adhesion of HT1080 cells to all 51 mutants. Results are shown as the percentage of HT1080 cell binding relative to native ICAM-4Fc used at 20 μg/mL (100%).