![Fig. 3. Activation of PAK. / (A) PAK autophosphorylation and kinase activity in stimulated platelets. PAK was immunoprecipitated from platelets stimulated as indicated for 3 minutes at 37°C and incubated with 1 μg MBP in the presence of 20 μM MgCl2-ATP and 0.185MBq (5 μCi) γ[32P]-ATP. Autophosphorylated PAK was visualized as a 65-kDa phosphoprotein, and its kinase activity was evaluated by phosphorylation of MBP (pMBP). The amount of immunoprecipitated PAK was controlled by anti-PAK1/2 immunoblotting. (B) Time course of PAK activation in thrombin-stimulated platelets. Platelets were stimulated for 0 to 3 minutes at 37°C, and PAK activation was evaluated as in vitro kinase activity toward MBP. Total PAK (arrow) was quantified by anti-PAK1/2 immunoblotting (left). Autophosphorylation of PAK in THR-stimulated platelets (1 minute) detected by direct immunoblotting with anti-phosphoPAK1/2 Thr423/Thr402 (pPAK; right). (C) Quantification of PAK activity during the time course of thrombin stimulation by densitometry analysis. Results are expressed as a ratio of pMBP to total PAK and reported as means ± SEM of 3 separate experiments (*P < .05). Controls with rabbit irrelevant IgG is shown in lane C.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/100/13/10.1182_blood.v100.13.4462/4/h82423531003.jpeg?Expires=1751070005&Signature=SfuffuBxykEIFbOb2B3ZBvqkcPJG1VS4v3G6H~fEgj5daDxCHcGQO7J7mUDzRwAJhly4oXDAPlyAB8HrZvxEOGGWLhrmAAj78uixZEhh1YVeaYngHSJq-St0jGUnL43DTos8u3cvddus3g8VFqD5nth7Q9JkR-glh5ahBlNXJJmOjTumomx9kllXgf1DD1l4nh0v4yDD~7jyoOdHEGlMjSh4BEcDLMUQW9GV3hObM3SVekrr7fc89nDX2nuoMZJRj8fGtT7gLP7PmskAj9ZPRCoj8NQxtatgW3bMwzimPjMWtEfFUlzkJ9y92qwvLPAszGxW2F7XQXy7IhOMaIrD6Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Activation of PAK.
(A) PAK autophosphorylation and kinase activity in stimulated platelets. PAK was immunoprecipitated from platelets stimulated as indicated for 3 minutes at 37°C and incubated with 1 μg MBP in the presence of 20 μM MgCl2-ATP and 0.185MBq (5 μCi) γ[32P]-ATP. Autophosphorylated PAK was visualized as a 65-kDa phosphoprotein, and its kinase activity was evaluated by phosphorylation of MBP (pMBP). The amount of immunoprecipitated PAK was controlled by anti-PAK1/2 immunoblotting. (B) Time course of PAK activation in thrombin-stimulated platelets. Platelets were stimulated for 0 to 3 minutes at 37°C, and PAK activation was evaluated as in vitro kinase activity toward MBP. Total PAK (arrow) was quantified by anti-PAK1/2 immunoblotting (left). Autophosphorylation of PAK in THR-stimulated platelets (1 minute) detected by direct immunoblotting with anti-phosphoPAK1/2 Thr423/Thr402 (pPAK; right). (C) Quantification of PAK activity during the time course of thrombin stimulation by densitometry analysis. Results are expressed as a ratio of pMBP to total PAK and reported as means ± SEM of 3 separate experiments (*P < .05). Controls with rabbit irrelevant IgG is shown in lane C.
