Tyrosine phosphorylation of total cell protein is not altered in WAS platelets. WASp-deficient platelets and control platelets were incubated in Tyrodes-HEPES buffer and stimulated by the addition of collagen (3 μg/mL) for 90 seconds and thrombin (1 U/mL) for 60 seconds and through cross-linking of FcγRIIA for 90 seconds with 1 μg/mL MoAb IV.3 for 60 seconds, followed by the addition of F(ab′)₂ (30 μg/mL). Stimulation was stopped by the addition of equal volume of 2× loading buffer. Proteins were separated by 10% SDS-PAGE, electroblotted onto PVDF membranes, and then immunoblotted using the antiphosphotyrosine MoAb 4G10. The same concentration of platelets (1 × 10⁸/mL) was used in samples from the WAS patients and controls. The gel is representative of 2 experiments.