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Blood, Vol. 113, Issue 2, 447-457, January 8, 2009
Group IVA cytosolic phospholipase A2 (cPLA2 ) and integrin IIbβ3 reinforce each other's functions during IIbβ3 signaling in platelets
Blood Prévost et al.
113: 447
Supplemental materials for: Prevost et al
Files in this Data Supplement:
- Figure S1. Recombinant integrin cytoplasmic tail mimics (JPG, 156 KB)
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All integrin cytoplasmic fusion proteins contain a 6-histidine tag (His6) and a biotinylation site for conjugation to neutravidin beads (Avi). β3 and αIIb cytoplasmic tails were fused to Fos and Jun heterodimerizing domains, respectively. Both Fos and Jun epitopes were flanked by a 5-amino acid flexible peptide spacer.
- Figure S2. cPLA2α recruitment and activation in CHO cells (JPG, 79.9 KB)
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(A) CHO cells stably expressing wild-type αIIbβ3, αIIbΔ996β3, or αIIbβ3Δ724 were transduced with lentivirus encoding either cPLA2α-HA or no cPLA2α-HA (Mock). After treatment with or without 1 mM MnCl2 + 300 µg/ml fibrinogen for 30 min at 37°C, cells were lysed and immunoprecipitations were carried out with anti-β3 antibody SSA6 or murine IgG1 as a control. Immunoprecipitates were assessed for cPLA2α activity as described in Materials and Methods. Data represent mean ± SEM of two independent experiments, each performed in triplicate. The presence of cPLA2α-HA and vimentin in αIIbβ3 immunoprecipitates was monitored by western blotting. (B) cPLA2α-HA and actin expression in cell lysates detected by western blotting.
- Figure S3. Interactions of cPLA2α−∕− platelets with fibrinogen (JPG, 147 KB)
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Platelets from cPLA2α−∕− mice and wild-type littermates were re-suspended to a final concentration of 5 × 107 platelets/ml in Walsh’s buffer and treated with CRP, PAR4 receptor-activating peptide or ADP for 15 min at 37°C in presence of 150 µg/ml Alexa Fluor 546-labeled fibrinogen. Binding of fibrinogen was assessed by flow cytometry. Results are representative of 4 individual experiments.
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