Association of WASp with Btk, PLCγ2, and Grb2. Lysate from resting or CRP (3 μg/mL)-stimulated platelets were incubated with GST linked to the SH3 domains of Btk (10 μg) and PLCγ2 (5 μg) and full-length Grb2 (10 μg). Proteins were separated on 10% SDS-PAGE and electroblotted to PVDF membranes. Membranes were immunoblotted using the antiphosphotyrosine MoAb 4G10 (upper panel). Membranes were stripped and reprobed with anti-WASp MoAb (lower panel). (A) Time course of WASp association to GST-PLCγ2-SH3. Several tyrosine phosphorylated proteins bind to GST-PLCγ2-SH3 but not to GST (90-second time point shown) from CRP-stimulated platelets. The 64-kD band was shown to contain WASp by stripping the blot and reprobing. (B) WASp association to GST-Btk-SH3. (C) WASp association to GST-Grb2. The gels are representative of 3 to 5 experiments.