Binding of α_EC and γC to the α_M I and α_X I domains.

(A) Different concentrations of ¹²⁵I-labeled ligands α_EC (●) and γC (▾) in TBS, 1 mM CaCl₂, and 1 mM MgCl₂ were added to wells coated with 10 μg/mL of the recombinant α_M I domain and incubated for 3 hours at 37°C. After washing with TBS and 0.05% Tween 20, bound radioactivity was measured and the amounts of bound ligands were calculated, with correction for nonspecific binding to BSA-coated wells. (B) Different concentrations of recombinant α_X I domain produced as a fusion protein with GST in TBS containing 1 mM MgCl₂, 1 mM CaCl₂, 0.05% Tween 20, and 5% glycerol were added to microtiter plates coated with 50 μg/mL α_EC (●) and γC (▾) and postcoated with 3% BSA. After incubation for 3 hours at 22°C, anti-GST mAb (1:5000) was added to the wells for an additional 1.5 hours. Binding of the I domains was then detected with a secondary goat anti–mouse IgG conjugated to alkaline phosphatase, with subsequent development of the reaction with p-nitrophenyl phosphate.