Distribution of SNAP-23 in platelets.

(A) Platelets were disrupted by freeze-thaw cycles and fractionated into a soluble fraction (S1) and pellet by ultracentrifugation. The pellet was incubated with 1 mol/L KCl on ice for 30 minutes. The supernatant (S2) was collected, and the resulting pellet was either solubilized with 1% Triton X-100 in PBS or washed again with 200 mmol/L Na₂CO₃ for 30 minutes. The supernatants (Triton-soluble, Stx; and carbonate-released, Sc) were collected and the pellets (Ptx and Pc) were resuspended in SDS-loading buffer. All of the above fractions were subjected to western blotting using ab23 and anti–syntaxin 4 antibody. Based on comparison with the starting material, approximately 38.2%, 16.5%, 33.5%, 31.1%, 14.2%, and 8.5% of the total platelet protein was in S1, S2, Sc, Stx, Ptx, and Pc, respectively. (B) The initial soluble and membrane fractions from A were incubated with 1% Triton X-114 for 30 minutes on ice. The aqueous and detergent phases were separated by warming the samples to 37°C, followed by centrifugation. The aqueous and detergent phases of both the soluble fraction and membrane pellet as well as the Triton X-114–insoluble pellet were analyzed by western blotting using ab23 and anti–syntaxin 4 antibody. His₆–SNAP-23 was also subjected to Triton X-114 partitioning, and the aqueous and detergent phases were analyzed by western blotting using ab23. (C) Soluble SNAP-23 is released from platelets after the treatment with 0.8 U/mL SLO. Platelets (10⁸) were treated with 0.8 U/mL SLO for 10 minutes and then pelleted by centrifugation. The supernatant (S) and the pellet (P) were subjected to western blotting with ab23 and anti–syntaxin 4.