Complex formation between LRP and α_Mβ₂. (Ai) Purified recombinant cluster IV (0-2.0 μM) was incubated with immobilized α_Mβ2 complex (2.5 μg/well) in Tris-buffered saline/3 mM CaCl₂/1 mM MnCl₂ (pH 7.4) for 2 hours at 37°C. Bound cluster IV was subsequently determined using peroxidase-labeled polyclonal antibodies directed against cluster IV. Data represent mean ± SEM of 4 experiments. (Aii) Binding of 200-nM cluster IV to immobilized α_Mβ2 in the presence or absence of a 10-fold excess of GST-RAP. (B) 500 nM purified recombinant I-domain of the α_M- (line I) or the α_L-subunit (line II) were perfused over LRP immobilized onto a CM5-sensor chip (7.7 fmol/mm2) in 100 mM NaCl, 0.005% Tween-20, 2 mM CaCl₂, 2 mM MnCl₂, 25 mM HEPES (pH 7.4) at a flow rate of 5 μL/min for 20 minutes at 25°C. Line III: Before perfusion, 500 nM α_M was preincubated with a 5-fold molar excess of fibrinogen for 30 minutes. Line IV: 5 μM fibrinogen. Line V: 500 nM GST. Ligand solution was replaced with buffer 10 minutes after injection to initiate dissociation. Depicted are sensorgrams corrected for aspecific binding, which was less than 5% of binding to LRP-coated channels.