Fig. 2. Lipid rafts can be isolated from the actin cytoskeleton of activated platelets. (A) Schematic representation of the isolation procedure of lipid rafts from the cytoskeleton of thrombin-activated platelets. A representative picture of the gradient obtained after centrifugation is shown. (B) Distribution of the raft marker proteins actin, CD36, LAT and in the eight fractions of the sucrose gradient containing the depolymerized actin cytoskeleton isolated from 3x10⁹ activated platelets (as described in A). Results are representative of two independent experiments. In the top panel, the distribution of actin was determined by densitometry analysis of western blots (10-15% of F-actin associated to lipid rafts). (C) Rafts were isolated from both the cytoskeleton fraction pre-treated with KI (0.6 M) and the supernatant (as described in A) prepared from resting or thrombin-activated platelets (3x10⁹ cells). The lipid composition of rafts (pooled fractions 2-4) was analyzed qualitatively and quantitatively. Results are expressed as nmoles of lipids and are the mean of two independent experiments with very similar results. Similar results were obtained in the presence or absence of 0.6 M of KI in the supernatant.