American Society of Hematology

Fig. 4.

$Fig. 4. Localization of human cellubrevin, SNAP-23, and syntaxin 2 by immunoblot analysis of subcellular fractions. / (A) Platelet cavitate was prepared as described in “Materials and methods,” and the fraction recovered from the 11% to 30% metrizamide interface was subjected to centrifugation at 100 000g for 1 hour. Supernatants and pellets were separated. Supernatants (sup) were subsequently lyophilized and solubilized in sample buffer. Proteins in the lyophilized supernatants and pellets were separated by SDS-PAGE and were analyzed for human cellubrevin, SNAP-23, and syntaxin 2 by immunoblotting with antihuman cellubrevin antibody, anti–SNAP-23 antibody, or antisyntaxin antibody, as indicated. (B) Platelet cavitate was prepared from biotin-labeled platelets and separated in a density step gradient of 11%, 15%, 20%, 25%, and 30% metrizamide. Fractions were analyzed using an avidin-HRP conjugate or by immunoblotting with anti-CD63 antibody, anti–P-selectin antibody, antihuman cellubrevin antibody, anti–SNAP-23 antibody, or antisyntaxin 2 antibody, as indicated.$

Localization of human cellubrevin, SNAP-23, and syntaxin 2 by immunoblot analysis of subcellular fractions.

(A) Platelet cavitate was prepared as described in “Materials and methods,” and the fraction recovered from the 11% to 30% metrizamide interface was subjected to centrifugation at 100 000g for 1 hour. Supernatants and pellets were separated. Supernatants (sup) were subsequently lyophilized and solubilized in sample buffer. Proteins in the lyophilized supernatants and pellets were separated by SDS-PAGE and were analyzed for human cellubrevin, SNAP-23, and syntaxin 2 by immunoblotting with antihuman cellubrevin antibody, anti–SNAP-23 antibody, or antisyntaxin antibody, as indicated. (B) Platelet cavitate was prepared from biotin-labeled platelets and separated in a density step gradient of 11%, 15%, 20%, 25%, and 30% metrizamide. Fractions were analyzed using an avidin-HRP conjugate or by immunoblotting with anti-CD63 antibody, anti–P-selectin antibody, antihuman cellubrevin antibody, anti–SNAP-23 antibody, or antisyntaxin 2 antibody, as indicated.

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