Role of PAK and Rac1 in Akt translocation. (A) Washed human platelets were stimulated for 1 minute at 37°C with 500 μM AYPGKF (AYP). Platelet lysates were immunoprecipitated (IP) with agarose-conjugated PAK rabbit polyclonal IgG, and samples were probed for coimmunoprecipitating Akt using anti-Akt mouse mAb (Cell Signaling Technology). (B) PAK1 knockout (KO) murine platelets were stimulated with 500 μM AYPGKF, and membrane fractions were subjected to SDS-PAGE. Western blots were then probed for anti-Akt, anti-phospho S473 Akt, and anti-PAK1 antibodies. β3-integrin was used as the lane loading control. (C) Washed human platelets were stimulated with AYPGKF (500 μM) in the presence or absence of 50 μM EHT1864, and the effect on PAK translocation and phosphorylation was evaluated using western blot analysis of platelet membrane fractions. β3-integrin was used as the lane loading control. The western blot analysis shown is representative of 3 independent experiments.
