SKAP-HOM protein expression and function in ADAP^+/+ or ADAP^−/− mice. (A) Washed platelets from ADAP^+/+ or ADAP^−/− mice were lysed, subjected to SDS gel electrophoresis, and Western blotted for SKAP-HOM. Blots were then reprobed for ADAP and integrin β3, the latter to monitor gel loading. Two separate anti–SKAP-HOM antibodies were used to confirm results. (B) SKAP-HOM regulation by ADAP in CHO cells. CHO cells were transfected with 1 μg ADAP alone, 3 μg EGFP-SKAP-HOM or EGFP-SKAP-HOM lacking the SH3 domain (dSH3) alone, or a combination of ADAP and EGFP-SKAP-HOM. After 48 hours, cells were subjected to SDS polyacrylamide gel electrophoresis and probed for ADAP and SKAP-HOM. Platelet lysate from wild-type mice, prepared as in Figure 6A, was used as a positive control, and blots were reprobed with a β-actin antibody to monitor gel loading. (C) Platelets from wild-type or SKAP-HOM knockout mice were allowed to adhere to dmA1A2 VWF as described in Figure 2, and integrin activation was assessed by FITC-fibrinogen binding in the presence or absence of a cocktail of inhibitors or PMA. Results in panels A and B are representative of at least 3 experiments, with similar results. Results in panel C are for ADAP^−/− platelets (white bars), depicted as a percentage of soluble ligand that is specifically bound by ADAP^+/+ platelets (black bars) ± SEM, and are a summary of 3 separate experiments.