Expression of Akt3 in platelets. (A) Human platelet RNA was isolated from washed platelets (3 × 10⁸). RT-PCR was performed with primers specific for Akt3 or a housekeeping gene, GAPDH. (B) Mouse platelet RNA was isolated from 3 × 10⁸ platelets of wild-type or Akt3^−/− platelets and RT-PCR was performed similarly. Leukocyte contamination of platelet preparation was 4 × 10⁴/mL as determined using Hemavet blood cell analyzer. RNA was isolated from 4 × 10⁴/mL of WT mouse leukocytes and was also analyzed by RT-PCR using Akt3 specific primers under the same conditions as for platelet preparations to verify that the Akt3 fragment was not from leukocyte contamination. (C) Washed human platelets, wild-type and Akt3^−/− mouse platelets were solubilized and immunoblotted with a rabbit antibody specifically recognizing Akt3, and α-tubulin is used as loading control. (D) Washed human platelets were solubilized, and immunoabsorbed with anti-Akt3 to remove Akt3 from lysates or with control rabbit IgG, and then immunoblotted with anti-Akt3 or an antibody recognizing all Akt isoforms (Total Akt). (E) Experiments in panel D were scanned and quantified using NIH Image J for uncalibrated optical density (mean ± SE, 4 experiments). The difference in percent of total Akt between IgG and Akt3 immunoabsorbed lysates is significant (P < .0125), as determined using paired t test.