FIGURE 5.
Efb interacts with platelet P-selectin. A, human platelets were ultrasonicated in the presence or absence of 3 mg/ml added fibrinogen. Sigma His-Select HF nickel affinity gel was used to capture His-tagged Efb- or Efb-N-interacting proteins from platelet lysates. Affinity precipitates were separated by SDS-PAGE and immunoblotted for integrin αIIb, P-selectin, multimerin-1, fibronectin, thrombospondin-1 (indicated by Thrombosp.-1), and His tag (to confirm Efb/Efb-N expression and immobilization). Immunoblots shown here are representative of those from three independent experiments. -ve, negative control; PD, pulldown. B, SPR data confirm the direct interaction of Efb and P-selectin. Recombinant P-selectin-Fc immobilized on COOHV-coated SensiQTM sensor chips was treated with increasing concentrations of Efb (100 nm (brown), 300 nm (purple), and 2.8 μm (green)). All experiments were performed using the SensiQTM Pioneer FE SPR platform (SensiQ Technologies). Experimental data were fit using the Qdat data analysis software. Curves were fitted using a least squares fitting algorithm. RU, response units.