Specific binding of HKa to HEK 293 cells transfected with Mac-1 (Mac-1–HEK 293).
FITC–HKa (at indicated concentrations) was incubated with HEK 293 cells either transfected with Mac-1 or control transfected in binding buffer containing 50 μmol/L ZnCl2 for 30 minutes at room temperature, as described in “Materials and methods.” The same reaction was also performed in the presence of 10 mmol/L EDTA to determine the nonspecific binding. Concentrations of FITC–HKa used were 0, 15, 30, 60, and 120 nmol/L. Reactions were carried out in wells of a filtration 96-well plate containing polyvinylidene difluoride membrane. After filtration, the fluorescence of cell-bound FITC–HKa was measured on a Cytofluor 2350 system. (•) HKa binding to Mac-1 transfected HEK 293 cells. (▾) HKa binding to untransfected HEK 293 cells. (○) HKa binding to Mac-1 transfected HEK 293 cells in the presence of 10 mmol/L EDTA. (▿) HKa binding to untransfected HEK 293 cells in the presence of 10 mmol/L EDTA. The data are the mean ± SE of triplicate reactions.
