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Blood, Vol. 107, Issue 8, 3145-3152, April 15, 2006
Arf6 plays an early role in platelet activation by collagen and convulxin
Blood Choi et al.
107: 3145
Supplemental materials for: Choi et al
Files in this Data Supplement:
- Figure S1 (JPG, 59.5 KB)
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(A) Resting or collagen (10 µg/mL)-stimulated platelets were lysed in 2× Rafts-Lysis buffer (50 mM MES (pH 6.5), 150 mM NaCl, 1% Triton X-100, 2× protease inhibitor cocktail), mixed with 80% sucrose solution, and layered into 15 mL ultracentrifuge tubes (Beckman Coulter, Fullerton, CA), with 30% and 5% sucrose layers added on top. Samples were centrifuged at 200,000 × g for 18 hrs, nine fractions (1 mL each) were collected, and proteins were concentrated using trichloroacetic acid (TCA) precipitation, followed by boiling with 2× SDS sample buffer. Equal amounts (15% of total lysates) were analyzed by western blot using anti-Arf6, anti-Srcp60, and anti-SNAP23. Pellet represents Triton X-100-insoluble cytoskeleton pellet and input represents 5% of total lysate. (B) Resting platelets were allowed to bind to collagen-coated coverslip, fixed, and stained with anti-Arf6 in the presence of 0.2% saponin and 10% FBS, followed by incubation with FITC-conjugated anti-rabbit antibody (Arf6) and TRITC-conjugated phalloidin (F-actin).
- Figure S2 (JPG, 59.9 KB)
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(A) Washed platelets (4 × 108/mL) were incubated with DMSO or myr-Arf6 peptide (25 µM) for 2 min with stirring at 37°C, followed by the stimulation with 0.1 µg/mL convulxin for the indicated times. The platelet suspensions were lysed and the supernatants were used to analyze Arf6-GTP. Five percent of the supernatants were analyzed by western blot as input controls. (B) Washed platelets were lysed and the supernatants were used to probe for Arf6-GTP using GST alone, GST-VHS, and GST-VHS-GAT. Western blot were performed using anti-Arf6 antibody. (C) Washed platelets were stimulated with indicated concentrations of convulxin. After incubation for the indicated times, platelets were lysed and the supernatant were analyzed for Arf6-GTP. (D) Washed platelets were incubated with DMSO (Resting and collagen) or myr-Arf6 peptide (25 µM) for 2 min with stirring at 37°C, followed by the stimulation with 10 µg/mL collagen. After incubation for the indicated times, platelets were lysed and the supernatant were analyzed for Arf1/3-GTP using GST-GGA3. (E) Washed platelets were incubated with 1% methanol (MeOH) or BFA (10 µg/mL) for 5 min with stirring at 37°C, followed by stimulation with 10 µg/mL collagen. Aggregation was monitored using aggregometer.
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