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Blood, Vol. 109, Issue 11, 4769-4776, June 1, 2007
Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids
Blood Malleier et al.
109: 4769
Supplemental materials for: Malleier et al, Vol 109, Issue 11, 4769-4776
Files in this Data Supplement:
- Figure S1. Effect of phospholipids on the interaction of aPC with PCI in the absence of Ca++ (PDF, 15 KB) -
aPC (5 nM) was incubated for 20 minutes with different concentration (as indicated) of recombinant PCI in the absence and presence of heparin (2.5 µg/mL) or phospholipids (as shown, 50 µg/mL each) in the absence of calcium in a buffer containing 5 mM EDTA. Remaining aPC activity was determined using the synthetic substrate S-2366 as described in “Materials and methods.” Data shown represent the means of duplicates. Similar results were obtained in at least 3 independent experiments performed with different preparations of recombinant PCI.
- Figure S2. The interfering effect of OxPAPE on aPC inhibition by PCI in the absence of Ca++ is dose dependent and can be reversed by heparin (PDF, 19.2 KB) -
(A) aPC (5 nM) was incubated for 20 minutes with PCI (90 nM) in the absence and presence of different concentrations of OxPAPE (as indicated) in the absence of calcium in a buffer containing 5 mM EDTA. Values shown are mean values from 2 determinations. (B) aPC (5 nM) was incubated for 20 minutes with PCI (90 nM) in the absence or presence of different concentrations of heparin (as shown) and/or OxPAPE (50 µg/mL) in the absence of calcium in a buffer containing 5 mM EDTA. Values shown are mean values from 2 determinations.
- Figure S3. Complex formation of aPC with PCI (A) and cleavage of PCI by aPC (B) in the absence of Ca++ (PDF, 24 KB) -
Complex formation of PCI with aPC and cleavage of PCI by aPC in the absence or presence of heparin, PAPE, or OxPAPE was analyzed by SDS-PAGE. aPC (200 nM) and PCI (400 nM) were incubated without or with heparin (1 µg/µL), PAPE (50 µg/mL), or OxPAPE (50 µg/mL) at 37°C (lanes 1-5). The reactions were stopped by addition of Laemmli buffer after 0 (lane 1) or 30 (lanes 2-5) minutes as indicated at the bottom of the figure and heating of the samples to 95°C. Incubation time 0 means that Laemmli buffer was added before aPC and PCI. aPC/PCI complex formation was analyzed on silver-stained gels (upper panel), and cleavage of PCI by aPCI on Western blots (lower panel), as described in “Materials and methods.”
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