Association of WASP, CrkL, Hic-5, and syk with the Triton X-100 insoluble residue.

Platelets were lysed with Triton X-100–EGTA buffer before or after stimulation with thrombin (1 U/mL), with or without stirring. Lysates were separated by high-speed centrifugation into soluble and insoluble residues. Proteins from each fraction were separated by 10% SDS-PAGE and immunoblotted with anti-WASP, anti-CrkL, anti-paxillin (cross-reactive with Hic-5), or anti-syk antibodies as indicated. Lane 1, Triton X-100 soluble residue of resting cells (7.5 × 10⁶ cells). Lane 2, Triton X-100 soluble residue of cells (7.5 × 10⁶ cells) prepared 10 minutes after exposure to thrombin (1 U/mL) with stirring. Lane 3, Triton X-100 soluble residue of cells (7.5 × 10⁶ cells), prepared 10 minutes after exposure to thrombin (1 U/mL) without stirring to prevent aggregation. Lane 4, Triton X-100 insoluble residue of resting cells (3.0 × 10⁷ cells). Lane 5, Triton X-100 insoluble residue from 3.0 × 10⁷ cells, prepared 10 minutes after exposure to thrombin (1 U/mL) with stirring. Lane 6, Triton X-100 insoluble residue from 3.0 × 10⁷ cells, prepared 10 minutes after exposure to thrombin (1 U/mL) without stirring.