Figure 2. STXBP5 is associated with platelet SNARE complexes and cytoskeleton.

(A–D) Platelet extracts (Input) from resting (R) or thrombin-stimulated (S) platelets were prepared by solubilization with 1% Triton X-100. After clarification, platelet extracts were incubated with anti-STXBP5 mAb (A) or rabbit polyclonal Ab (B and D), anti-STX11 rabbit polyclonal Ab (C), anti-Munc18b goat polyclonal Ab (D), or IgG control for 3 hours at 4°C. Immune complexes were recovered with protein A and G sepharose. The bound proteins were eluted and separated by SDS-PAGE, followed by IB with the indicated antibodies. (E) Washed platelets (2.5 × 10⁸) were resuspended in HEPES/Tyrode buffer and incubated with (stimulated) or without (resting) 0.1 U/ml thrombin for 5 minutes. After disruption by freeze-thaw, the unbroken cells were removed by centrifugation at 700 g for 5 minutes, and supernatants were subjected to ultracentrifugation (100,000 g for 1 hour at 4°C). The cytosolic fractions (S1) were collected. The remaining pellets were solubilized sequentially with Triton X-100 (S2), then n-octyl-β-d-glucopyranoside (S3). The supernatants and insoluble pellet (P) were analyzed by IB with the indicated antibodies. T, total extract (starting material for the fractionation).