Binding of PCI to immobilized and soluble phospholipids. (A) Phospholipids (100 μg/mL) were immobilized on microtiter plates (Nunc maxisorp; Nunc) and incubated with different concentrations of recombinant PCI (as indicated). Bound PCI was detected as described in “Materials and methods.” Absorbance values shown are corrected for nonspecific binding to uncoated plates and represent means ± SEM of triplicates. The absorbance values at 405 nm for nonspecific binding were between 0.130 and 0.215. Similar results were obtained in at least 3 independent experiments. (B) Microtiter plates were coated with OxPAPE (100 μg/mL) and incubated with 4 nM recombinant PCI in the absence or presence of phospholipids (as indicated, 100 μg/mL each), retinoic acid (5 μM), or annexin V (40 nM in a buffer containing 2.5 mM Ca⁺⁺). Bound PCI was detected as described in “Materials and methods.” Means of triplicates ± SEM are shown, *P ≤ .001. (C) PCI (350 nM) was incubated with PAPE, OxPAPE, or OxPAPS (100 μg/mL each) as described in “Materials and methods” and loaded on a native 8% PAGE gel. Western blotting was done as described in “Materials and methods.” (D) PAPS, OxPAPS (i), or OxPAPE (ii) (100 μg/mL each) was immobilized on microtiter plates and incubated with different concentrations of recombinant PCI. Bound PCI was detected as described in “Materials and methods.” Means of triplicates ± SEM are shown. (E) Phospholipids were immobilized on microtiter plates (as indicated, 100 μg/mL each) and incubated with PCI (4 nM) in the absence and presence of heparin (concentrations as indicated). Bound PCI was detected as described in “Materials and methods.” Means of triplicates ± SEM are shown.