CSPα is membrane associated but not present in lipid rafts. (A) Lysates were prepared from washed human platelets subjected to 5 freeze-thaw cycles and centrifuged to separate membrane and cytosol (S1) fractions. The membrane fraction was treated with 1% Triton X-100 to generate Triton X-100–soluble (S_TX) and Triton X-100–insoluble (I_TX) fractions, which were separated by ultracentrifugation. The fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and probed by western blotting with the indicated antibodies. Data are representative of 2 independent experiments and quantification is based on the mean of the representative experiment. (B) Resting or 0.1 U/mL thrombin–stimulated human platelets were lysed with 2× lysis buffer and layered under a sucrose gradient. The samples were centrifuged and collected in 1-mL fractions. The fractions were analyzed by SDS-PAGE and probed by western blotting for the indicated antibodies syntaxin-11 and CSPα. Data are representative of 2 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; R, resting; S, stimulated.